OVEREXPRESSION OF AMINOACYL-tRNA SYNTHETASES FOR EFFICIENT PRODUCTION OF ENGINEERED PROTEINS CONTAINING AMINO ACID ANALOGUES

ABSTRACT

Methods for producing modified polypeptides containing amino acid analogues are disclosed. The invention further provides purified dihydrofolate reductase polypeptides, produced by the methods of the invention, in which the methionine residues have been replaced with homoallylglycine, homoproparglycine, norvaline, norleucine, cis-crotylglycine, trans-crotylglycine, 2-aminoheptanoic acid, 2-butynylglycine and allylglycine.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of allowed U.S. patent applicationSer. No. 10/612,713, filed Jul. 1, 2003, which is a continuation of U.S.patent application Ser. No. 09/767,515, filed Jan. 23, 2001, (now U.S.Pat. No. 6,586,207), which application claims the benefit under 35U.S.C. § 119(e) of U.S. Provisional Application No. 60/207,627, filedMay 26, 2000, where these two applications are incorporated herein byreference in their entireties.

STATEMENT OF GOVERNMENT INTEREST

This invention was made with Government support under NSF Grant Nos. NSFDMR-9996048 and US Army Research Grant DAAG55-98-1-0518. The Governmenthas certain rights in this invention.

Throughout this application various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to novel compositions and methods, forincorporating amino acid analogues into proteins in vivo, byoverexpression of aminoacyl-tRNA synthetases.

2. Description of the Related Art

Expanding the scope of biological polymerizations to include non-naturalmonomers is an area of growing interest, with important theoretical andpractical consequences. An early and critically important example ofsuch studies was the demonstration that “dideoxy” nucleotide monomerscan serve as substrates for DNA polymerases. Advances in DNA sequencing(F. Sanger, S, Nicklen, A. R. Coulson, Proc. Natl. Acad. Sci. USA 1977,74, 5463-5467), DNA base pairing models (M. J. Lutz, S. A. Benner, S.Hein, G. Breipohl, E. Uhlmann, J. Am. Chem. Soc. 1997, 119, 3177-3178;J. C. Morales, E. T. Kool, Nature Struct. Biol. 1998, 5, 950-954),materials synthesis (W. H. Park, R. W. Lenz, S. Goodwin, Macromolecules1998, 31, 1480-1486; Y. Doi, S. Kitamura, H. Abe, Macromolecules 1995,28, 4822-4828), and cell surface engineering (K. J. Yarema, L. K. Mahal,R. E. Bruehl, E. C. Rodriguez, C. R. Bertozzi, J. Biol. Chem. 1998, 273,31168-31179; L. K. Mahal, K. J. Yarema, C. R. Bertozzi, Science 1997,276, 1125-1128; Saxon, E. and Bertozzi, C. R. Science 2000, 287,2007-2010) have resulted from the recognition of non-natural monomers bythe enzymes that control these polymerizations.

Recent investigations have shown the incorporation of modified orcompletely “synthetic” bases into nucleic acids (Matray, T. J.; Kool, E.T. Nature 1999, 399, 704; Kool, E. T. Biopolymers 1998, 48, 3; Morales,J. C.; Kool, E. T. Nature Struct. Biol. 1998, 5, 950; Guckian, K. M.;Kool, E. T.; Angew. Chem. Int. Ed. Eng. 1998, 36, 2825; Liu, D. Y.;Moran, S.; Kool, E. T. Chem. Biol. 1997, 4, 919; Moran, S.; Ren, R. X.F.; Kool, E. T. Proc. Natl. Acad. Sci. USA 1997, 94, 10506; Moran, S. etal. J. Am. Chem. Soc. 1997, 119, 2056; Benner, S. A. et al. Pure Appl.Chem. 1998, 70, 263; Lutz, M. J.; Horlacher J.; Benner, S. A. Bioorg.Med. Chem. Lett. 1998, 8, 1149; Lutz, M. J.; Held, H. A.; Hottiger, M.;Hubscher, U.; Benner, S. A. Nuc. Acids Res. 1996, 24, 1308; Horlacher,J. et al. Proc. Natl. Acad. Sci. USA 1995, 92, 6329; Switzer, C. Y.;Moroney, S. E.; Benner, S. A. Biochemistry 1993, 32, 10489; Lutz, M. J.;Horlacher, J.; Benner, S. A. Bioorg. Med. Chem. Lett. 1998, 8, 499;Switzer, C.; Moroney, S. E.; Benner, S. A. J. Am. Chem. Soc. 1989, 111,8322; Piccirilli, J. A.; Krauch, T.; Moroney, S. E.; Benner, S. A.Nature 1990, 343, 33), while materials researchers have exploited thebroad substrate range of the poly(β-hydroxyalkanoate) (PHA) synthases toprepare novel poly(β-hydroxyalkanoate)s (PHAs) with unusual physicalproperties (Kim, Y. B.; Rhee, Y. H.; Lenz, R. W. Polym. J. 1997, 29,894; Hazer, B.; Lenz, R. W.; Fuller, R. C. Polymer 1996, 37, 5951; Lenz,R. W.; Kim, Y. B.; Fuller, R. C. FEMS Microbiol. Rev. 1992, 103, 207;Park, W. H.; Lenz, R. W.; Goodwin, S. Macromolecules 1998, 31, 1480;Ballistreri, A. et al. Macromolecules 1995, 28, 3664; Doi, Y.; Kitamura,S.; Abe, H. Macromolecules 1995, 28, 4822).

Novel polymeric materials with unusual physical and/or chemicalproperties are also useful in polymer chemistry. The last severaldecades have shown many advances in synthetic polymer chemistry thatprovide the polymer chemist with increasing control over the structureof macromolecules (Szwarc, M. Nature 1956, 178, 1168-1169 Szwarc, M.Nature 1956, 178, 1168-1169; Faust, R.; Kennedy, J. P. Polym. Bull.1986, 15, 317-323; Schrock, R. R. Acc. Chem. Res. 1990, 23, 158-165;Corradini, P. Macromol. Symp. 1995, 89, 1-11; Brintzinger, H. H.;Fischer, D.; Mulhaupt, R.; Rieger, B.; Waymouth, R. M. Angew. Chem. Int.Ed. Engl. 1995, 34, 1143-1170; Dias, E. L.; SonBinh, T. N.; Grubbs, R.H. J. Am. Chem. Soc. 1997, 119, 3887-3897; Chiefari, J. et al.Macromolecules 1998, 31, 5559-5562). However, none of these methods haveprovided the level of control that is the basis of the exquisitecatalytic, informational, and signal transduction capabilities ofproteins and nucleic acids (Ibba, M.; Soll, D. Science 1999, 286,1893-1897). There remains a need for control over protein synthesis todesign and produce artificial proteins having advantageous properties.

For this reason, the design and synthesis of artificial proteins thatexhibit novel and potentially useful structural properties have beeninvestigated. Harnessing the molecular weight and sequence controlprovided by in vivo synthesis would permit control of folding,functional group placement, and self-assembly at the angstrom lengthscale. Proteins that have been produced by in vivo methods exhibitpredictable chain-folded lamellar architectures (Krejchi, M. T.; Atkins,E. D. T.; Waddon, A. J.; Fournier, M. J.; Mason, T. L.; Tirrell, D. A.Science 1994, 265, 1427-1432; Parkhe, A. D.; Fournier, M. J.; Mason, T.L.; Tirrell, D. A. Macromolecules 1993, 26(24), 6691-6693; McGrath, K.P.; Fournier, M. J.; Mason, T. L.; Tirrell, D. A. J. Am. Chem. Soc.1992, 114, 727-733; Creel, H. S.; Fournier, M. J.; Mason, T. L.;Tirrell, D. A. Macromolecules 1991, 24, 1213-1214), unique smecticliquid-crystalline structures with precise layer spacings (Yu, S. M.;Conticello, V.; Zhang, G.; Kayser, C.; Fournier, M. J.; Mason, T. L.;Tirrell, D. A. Nature 1997, 389, 187-190), and controlled reversiblegelation (Petka, W. A.; Hardin, J. L.; McGrath, K. P.; Wirtz, D.;Tirrell, D. A. Science 1998, 281, 389-392). The demonstrated ability ofthese protein polymers to form unique macromolecular architectures willbe of importance for engineering materials with interestingliquid-crystalline, crystalline, surface, electronic, and opticalproperties.

Novel chemical and physical properties that can be engineered intoprotein polymers may be expanded by the precise placement of amino acidanalogues. Efforts to incorporate novel amino acids into proteins invivo have relied on the ability of the translational apparatus torecognize amino acid analogues that differ in structure andfunctionality from the natural amino acids. The in vivo incorporation ofamino acid analogues into proteins is controlled most stringently by theaminoacyl-tRNA synthetases (AARS), the class of enzymes that safeguardsthe fidelity of amino acid incorporation into proteins (FIG. 1). The DNAmessage is translated into an amino acid sequence via the pairing of thecodon of the messenger RNA (mRNA) with the complementary anticodon ofthe aminoacyl-tRNA. Aminoacyl-tRNA synthetases control the fidelity ofamino acid attachment to the tRNA. The discriminatory power of theaminoacyl-tRNA synthetase places severe limits on the set of amino acidstructures that can be exploited in the engineering of natural andartificial proteins in vivo.

Several strategies for circumventing the specificity of the synthetaseshave been explored. Introduction of amino acid analogues can be achievedrelatively simply via solid-phase peptide synthesis (Merrifield, R. B.Pure & Appl. Chem. 1978, 50, 643-653). While this method circumvents allbiosynthetic machinery, the multistep procedure is limited to synthesisof peptides less than or equal to approximately 50 amino acids inlength, and is therefore not suitable for producing protein materials oflonger amino acid sequences.

Chemical aminoacylation methods, introduced by Hecht and coworkers(Hecht, S. M. Acc. Chem. Res. 1992, 25, 545; Heckler, T. G.; Roesser, J.R.; Xu, C.; Chang, P.; Hecht, S. M. Biochemistry 1988, 27, 7254; Hecht,S. M.; Alford, B. L.; Kuroda, Y.; Kitano, S. J. Biol. Chem. 1978, 253,4517) and exploited by Schultz, Chamberlin, Dougherty and others(Cornish, V. W.; Mendel, D.; Schultz, P. G. Angew. Chem. Int. Ed. Engl.1995, 34, 621; Robertson, S. A.; Ellman, J. A.; Schultz, P. G. J. Am.Chem. Soc. 1991, 113, 2722; Noren, C. J.; Anthony-Cahill, S. J.;Griffith, M. C.; Schultz, P. G. Science 1989, 244, 182; Bain, J. D.;Glabe, C. G.; Dix, T. A.; Chamberlin, A. R. J. Am. Chem. Soc. 1989, 111,8013; Bain, J. D. et al. Nature 1992, 356, 537; Gallivan, J. P.; Lester,H. A.; Dougherty, D. A. Chem. Biol. 1997, 4, 740; Turcatti, et al. J.Biol. Chem. 1996, 271, 19991; Nowak, M. W. et al. Science, 1995, 268,439; Saks, M. E. et al. J. Biol. Chem. 1996, 271, 23169; Hohsaka, T. etal. J. Am. Chem. Soc. 1999, 121, 34), avoid the synthetases altogether,but provide low protein yields.

Alteration of the synthetase activities of the cell is also possible,either through mutagenesis or through introduction of heterologoussynthetases (Ibba, M.; Hennecke, H. FEBS Lett. 1995, 364, 272; Liu, D.R.; Maghery, T. J.; Pastrnak, M.; Schultz, P. G. Proc. Natl. Acad. Sci.USA, 1997, 94, 10092; Furter, R. Protein Sci. 1998, 7, 419; Ohno, S. etal., J. Biochem. 1998, 124, 1065; Liu, D. R.; Schultz, P. G. Proc. Natl.Acad. Sci. 1999, 96, 4780; Wang, L.; Magliery, T. J.; Liu, D. R.;Schultz, P. G. J. Am. Chem. Soc. 2000, 122, 5010-5011; Pastrnak, M.;Magliery, T. J.; Schultz, P. G. Helv. Chim. Acta 2000, 83, 2277-2286).

In some instances, the ability of the wild-type synthetases to acceptamino acid analogues has been exploited. For example, wild-typesynthetases have been shown to activate and charge substrates other thanthe canonical, proteinogenic amino acids (Cowie, D. B.; Cohen, G. N.Biochim. Biophys. Acta. 1957, 26, 252; Richmond, M. H. Bacteriol Rev.1962, 26, 398; Horton, G.; Boime, I. Methods Enzymol. 1983, 96, 777;Wilson, M. J.; Hatfield, D. L. Biochim. Biophys. Acta 1984, 781, 205).This approach offers important advantages with respect to syntheticefficiency, in that neither chemical acylation of tRNA nor cell-freetranslation is required. The simplicity of the in vivo approach, itsrelatively high synthetic efficiency, and its capacity for multisitesubstitution, make it the method of choice for production of proteinmaterials whenever possible.

The capacity of the wild-type translational apparatus has beenpreviously demonstrated to utilize amino acid analogues bearingfluorinated (Richmond, M. H. J. Mol. Biol. 1963, 6, 284; Fenster, E. D.;Anker, H. S. Biochemistry 1969, 8, 268; Yoshikawa, E.; Fournier, M. J.;Mason, T. L.; Tirrell, D. A. Macromolecules 1994, 27, 5471), unsaturated(Van Hest, J. C. M.; Tirrell, D. A. FEBS Lett. 1998, 428, 68; Deming, T.J.; Fournier, M. J.; Mason, T. L.; Tirrell, D. A. J. Macromol. Sci.—PureAppl. Chem. 1997, A34, 2134), electroactive (Kothakota, S.; Mason, T.L.; Tirrell, D. A.; Fournier, M. J. J. Am. Chem. Soc. 1995, 117, 536),and other useful side chain functions. The chemistries of the abovefunctional groups are distinct from the chemistries of the amine,hydroxyl, thiol, and carboxylic acid functional groups characteristic ofproteins; this makes their incorporation particularly attractive fortargeted chemical modification of proteins.

For example, alkene functionality introduced into artificial proteinsvia dehydroproline can be quantitatively modified via bromination andhydroxylation (Deming, T. J.; Fournier, M. J.; Mason, T. L.; Tirrell, D.A. J. Macromol. Sci. Pure Appl. Chem. 1997, A34, 2143-2150). Alkenefunctionality, introduced by incorporation of other amino acidanalogues, should be useful for chemical modification of proteins byolefin metathesis (Clark, T. D.; Kobayashi, K.; Ghadiri, M. R. Chem.Eur. J. 1999, 5, 782-792; Blackwell, H. E.; Grubbs, R. H. Angew. Chem.Int. Ed. Engl. 1998, 37, 3281-3284), palladium-catalyzed coupling(Amatore, C.; Jutand, A. J. Organomet. Chem. 1999, 576, 255-277; Tsuji,J. Palladium Reagents and Catalysts: Innovations in Organic Synthesis;John Wiley and Sons: New York, 1995; Schoenberg, A.; Heck, R. F. J. Org.Chem. 1974, 39, 3327-3331), and other chemistries (Trost, B. M.;Fleming, I., Eds. Comprehensive Organic Synthesis; Pergamon Press:Oxford, 1991). The incorporation of fluorinated functional groups intoproteins has imparted to protein films the low surface energycharacteristic of fluoropolymers; contact angles of hexadecane onfluorinated protein polymers (70°) are much higher than, those onunfluorinated controls (17°) (Yoshikawa, E.; Fournier, M. J.; Mason, T.L.; Tirrell, D. A. Macromolecules 1994, 27, 5471-5475).

Methionine (1) (FIG. 1) is a possible target for substitution by aminoacid analogues, with its hydrophobicity and polarizability, make it animportant amino acid for regulating protein structure andprotein-protein recognition processes (T. Yuan, A. M. Weljie, H. J.Vogel, Biochemistry 1998, 37, 3187-3195; H. L. Schenck, G. P. Dado, S.H. Gellman, J. Am. Chem. Soc. 1996, 118, 12487-12494; Maier, K. L.;Lenz, A. G., Beck-Speier, I.; Costabel, U. Methods Enzymol. 1995, 251,455-461). Replacement of methionine by its analogues may thereforepermit purposeful manipulation of these properties.

Several analogues of methionine (1), specifically selenomethionine,telluromethionine, norleucine, trifluoromethionine and ethionine(Hendrickson, W. A.; Horton, J. R.; Lemaster, D. M. EMBO J. 1990, 9,1665; Boles, J. O. et al. Nature Struct. Biol. 1994, 1, 283; Cowie, D.B.; Cohen, G. N.; Bolton, E. T.; de Robichon-Szulmajster, H. Biochim.Biophys. Acta 1959, 34, 39; Duewel, H.; Daub, E.; Robinson, R.; Honek,J. F. Biochemistry 1997, 36, 3404; Budisa, N.; Steipe, B.; Demange, P.;Eckerskorn, C.; Kellerman, J.; Huber, R. Eur. J. Biochem. 1995, 230,788), have been shown to exhibit translational activity in bacterialhosts. Incorporation of selenomethionine in place of methionine has longbeen known to facilitate protein structure determination by x-raycrystallography (Wei, Y.; Hendrickson, W. A.; Crouch, R. J.; Satow, Y.Science 1990, 249, 1398-1405).

However, only a limited number of amino acid analogues have been shownto conclusively exhibit translational activity in vivo, and the range ofchemical functionality accessible via this route remains modest. Thesecircumstances dictate a need for a systematic search for new amino acidanalogues and strategies that will allow the engineering of proteinswith novel chemical and physical properties.

BRIEF SUMMARY OF THE INVENTION

The present invention seeks to overcome these and other disadvantages inthe prior art by providing a novel method for incorporating amino acidanalogues into polypeptides of interest in vivo by expanding the scopeof amino acid analogues that are incorporated and increasing proteinyields. Preferably, the production of modified polypeptides can be in ahost-vector system in which a natural amino acid in the wild-typepolypeptide is replaced with a selected amino acid analogue byoverexpressing an aminoacyl-tRNA synthetase corresponding to the naturalamino acid so replaced.

In addition, the present invention provides novel host-vector systems.The host-vector system produces an aminoacyl-tRNA synthetase in anamount in excess of the level of a naturally occurring aminoacyl-tRNAsynthetase. The system also produces a polypeptide of interest in anamount in excess of the level produced by a naturally occurring geneencoding the polypeptide of interest.

Nucleic acids encoding the expression vectors, hosts, and methods ofintegrating a desired amino acid analogue into target polypeptides arealso provided.

The invention further provides purified dihydrofolate reductasepolypeptides, produced by the methods of the invention, in which themethionine residues have been replaced with homopropargylglycine(2-amino-hexynoic acid), homoallylglycine (2-amino-hexenoic acid),cis-crotylglycine (cis-2-amino-4-hexenoic acid), trans-crotylglycine(trans-2-amino-4-hexenoic acid), norleucine, 6,6,6-trifluoro-2-aminohexanoic acid, 2-amino-heptanoic acid, norvaline, o-allylserine,2-butynylglycine, allylglycine or propargylglycine. The formation of themodified polypeptides demonstrate the ease and efficiency of the methodsof the invention for incorporating amino acid analogues such as,methionine analogues, into proteins such as, dihydrofolate reductase.

Using the methods of the invention, it is possible to produce entirelynew polypeptides containing amino acid analogues having unusualproperties.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

FIG. 1 depicts a schematic diagram of in vivo protein synthesis.

FIG. 2 depicts a set of methionine analogues (2-13), as described inExample 1, infra.

FIG. 3 illustrates the SDS-PAGE analysis of mDHFR synthesis by E. colistrain CAG18491/pREP4/pQE15, as described in Example I, infra. Cultureswere supplemented with methionine or with one of the analogues 2-9, asindicated. Each lane is identified in terms of the time of analysissubsequent to addition of the inducer IPTG. mDHFR is visualized bystaining with Coomassie Brilliant Blue. The target protein can bedetected only in cultures supplemented with methionine or with analogues2, 3, or 9, respectively.

FIG. 4 shows the determination of the occupancy of the initiator sitein: a). mDHFR, b). mDHFR-E (alkene) and c). mDHFR-Y (alkyne), asdescribed in Example I, infra. Chromatograms are shown for analysis ofthe N-terminal residue in each of the three proteins, as determined viaEdman degradation. The signals corresponding to methionine, 2 and 3elute at 12.3, 14.3 and 11.0 min, respectively. The strong signal at13.8 min is due to piperidylphenylthiourea, a by-product of theanalysis. Signals assigned to 2 and 3 were verified by analysis ofauthentic samples of the analogues.

FIG. 5 depicts the activation of methionine and methionine analogues byMetRS (Methionyl tRNA synthetase), as described in Example I, infra. Theamount of PP_(i) exchanged in 20 minutes is shown for methionine (1) andfor methionine analogues 2-13. The background (14) is given for areaction mixture lacking both enzyme and amino acid.

FIG. 6 illustrates the electron density maps (colored surfaces) andnegative isopotential surfaces (meshes) for methionine (a) and foranalogues 2, 3 and 5 (b-d, respectively), as described in Example I,infra. The electron density maps indicate electron-rich (red) andelectron-poor (blue) regions of each molecule. For simplicity, the aminoacid form is shown; this avoids representation of the highly extendedisopotential surface of the carboxylate anion of the zwitterion andfacilitates comparison of side-chain electronic structure.

FIG. 7 shows the SDS-PAGE analysis of mDHFR synthesis by E. coli strainsB834(DE3)/pQE15/pREP4 (designated pQE15) and B834(DE3)/pQE15-MRS/pREP4(designated pQE15-MRS), as described, in Example II, infra. Cultures(M9+19AA) were supplemented with nothing (−Met), methionine (Met) ortrans-crotylglycine (60 mg/L) (Tcg), as indicated.

FIG. 8 depicts the SDS-PAGE analysis of DHFR synthesis by E. colistrains CAG18491/pQE15/pREP4 and CAG18491/pQE15-MRS/pREP4, as describedin Example II, infra. Cultures (M9+19AA) were supplemented with nothing(−Met), methionine (+Met), or 2-butynylglycine (60 mg/L) (+2bg), asindicated.

FIG. 9 shows the SDS-PAGE analysis of mDHFR synthesis by E. coli strainsCAG18491/pQE15/pREP4 (the left panel in each pair, PQE15) andCAG18491/pQE15-MRS/pREP4 (the right panel in each pair, pQE15-MRS), asdescribed in Example II, infra. Cultures (M9+19AA) were supplementedwith the analogues 4, 6, 7, 8, 10, 12, and 13 at 500 mg/l, as indicated.Negative (−Met) and positive (+Met) controls of CAG18491/pQE15-MRS/pREP4cultures are also shown for comparison

FIG. 10 is a table detailing kinetic parameters for methionine analoguesin the ATP-PP_(i) exchange reaction and analogue's ability to supportprotein biosynthesis in cultures of a conventional bacterial hostsupplemented with the analogues, as described in Example II, infra.

FIG. 11 illustrates the activation rates of methionine by whole celllysates, as described in Example II, infra. Maximum ATP-PP_(i) exchangevelocities, measured at a saturating concentration of methionine (750μM), are shown for whole cell lysates of B834(DE3)/pQE15/pREP4 (solid)and of B834(DE3)/pQE15-MRS/pREP4 (striped). Rates were measured for (a)cell lysates obtained from cultures prior to protein expression, (b)cell lysates obtained from cultures supplemented with methionine duringprotein expression, and (c) cell lysates obtained from culturessupplemented with transcrotylglycine during protein expression.

FIG. 12 shows the Proton NMR spectra (599.69 MHz) of (a) mDHFR, (b) Tcg,(c and d) mDHFR-Tcg, as described in Example II, infra. Samples weredissolved at concentrations of approximately 10 mg/ml in D₂O containing2% d-formic-d-acid and spectra were collected at 25° C. overnight.

FIG. 13 depicts the N-terminal sequencing results indicating occupancyof the initiator site in mDHFR-Tcg, as described in Example II, infra.Chromatograms are shown for (a) the N-terminal residue of mDHFR, (b) Tcgcontrol, and (c) the N-terminal residue of mDHFR-Tcg, as determined viaEdman degradation.

FIG. 14 depicts the N-terminal sequencing results indicating occupancyof the initiator site in mDHFR-bg, as described in Example II, infra.Chromatograms are shown for (a) the N-terminal residue of mDHFR, (b) 2bgcontrol, and (c) the N-terminal residue of mDHFR-2bg, as determined viaEdman degradation.

FIG. 15 is a table of the kinetic parameters for methionine analogues inthe ATP-PP_(i) exchange reaction and protein yields for bacterialcultures supplemented with the analogues, as described in Example III,infra.

FIG. 16 shows the comparison of the kinetic parameters for methionineanalogues in the ATP-PP_(i) exchange reaction and relative proteinyields from conventional bacterial host cultures supplemented with theanalogues, as described in Example III, infra.

FIG. 17 depicts the Western blot analysis of protein synthesis bybacterial expression hosts CAG18491/pQE15/pREP4 (pQE15) andCAG18491/pQE15-MRS/pREP4 (MRS). Bacterial cultures were supplementedwith methionine, 2, 3 or 9, as described in Example III, infra.

FIG. 18 illustrates the activation (a) and aminoacylation (b) steps ofamino acid attachment to tRNA, as described in Example III, infra.

FIG. 19 depicts the sequence of pQE15-MRS (SEQ ID NO.: 1).

FIG. 20 depicts the sequence of pQE15-W305F (SEQ ID NO.: 2).

DETAILED DESCRIPTION OF THE INVENTION

As used in this application, the following words or phrases have themeanings specified.

Definitions

As used herein, a polypeptide refers to a peptide or protein havingnatural amino acids.

As used herein, modified polypeptides are polypeptides having amino acidanalogues incorporated into their amino acid sequence.

As used herein, a “natural amino acid” is one of the 20 naturallyoccurring amino acids, namely glycine, alanine, valine, leucine,isoleucine, serine, threonine, aspartic acid, glutamic acid, asparagine,glutamine, lysine, arginine, cysteine, methionine, phenylalanine,tyrosine, tryptophan, histidine and proline.

As used herein, the term “amino acid analogue” refers to a compound thathas a structure analogue to a natural amino acid but mimics thestructure and/or reactivity of a natural amino acid. This includes allamino acids but the natural 20 amino acids are referred to as amino acidanalogues even if they are naturally present (e.g., hydroxyproline).

As used herein, the term “peptide” refers to a class of compoundscomposed of amino acids chemically bound together with amide linkages(CONH). Peptide as used herein includes oligomers of amino acids andsmall and large peptides, including polypeptides.

As used herein, “polypeptides” embrace all peptides and thosepolypeptides generally defined as proteins and also those that areglycosylated, e.g., glycoproteins.

METHODS OF THE INVENTION

The present invention is based on the discovery that incorporation ofamino acid analogues into polypeptides can be improved in cells thatoverexpress aminoacyl-tRNA synthetases that recognize amino acidanalogues as substrates. “Improvement” is defined as either increasingthe scope of amino acid analogues (i.e., kinds of amino acid analogues)that are incorporated or by increasing the yield of the modifiedpolypeptide. Overexpression of the aminoacyl-tRNA synthetase increasesthe level of aminoacyl-tRNA synthetase activity in the cell. Theincreased activity leads to an increased rate of incorporation of aminoacid analogues into the growing peptide, thus the increased rate ofsynthesis of the polypeptides, thereby increasing the quantity ofpolypeptides containing amino acid analogues, i.e., modifiedpolypeptides, produced.

In general, the methods of the invention comprises introducing into ahost cell, a vector having nucleic acids encoding an aminoacyl-tRNAsynthetase, and nucleic acids encoding a polypeptide of interest toproduce a host-vector system. The nucleic acids, encoding theaminoacyl-tRNA synthetase, and the nucleic acids encoding thepolypeptide of interest, may be located in the same or differentvectors. The vectors include expression control elements which directthe production of the aminoacyl-tRNA synthetase, and the polypeptide ofinterest. The expression control elements (i.e., regulatory sequences)can include inducible promotors, constitutive promoters, secretionsignals, enhancers, transcription terminators, and other transcriptionalregulatory elements.

In the host-vector system, the production of an aminoacyl-tRNAsynthetase can be controlled by a vector which comprises expressioncontrol elements that direct the production of the aminoacyl-tRNAsynthetase. Preferably, the production of aminoacyl-tRNA synthetase isin an amount in excess of the level of naturally occurringaminoacyl-tRNA synthetase, such that the activity of the aminoacyl-tRNAsynthetase is greater than naturally occurring levels.

In the host-vector system, the production of a polypeptide of interestcan be controlled by a vector which comprises expression controlelements for producing the polypeptide of interest. Preferably, thepolypeptide of interest so produced is in an amount in excess of thelevel produced by a naturally occurring gene encoding the polypeptide ofinterest.

The host-vector system can be constitutively overexpressing theaminoacyl-tRNA synthetase and induced to overexpress the polypeptide ofinterest by contacting the host-vector system with an inducer, such asisopropyl-β-D-thiogalactopyranoside (IPTG). The host-vector system canalso be induced to overexpress the aminoacyl-tRNA synthetase and/or theprotein of interest by contacting the host-vector system with aninducer, such as IPTG. Other inducers include stimulation by an externalstimulation such as heat shock.

Using the methods of the invention, any natural amino acid can beselected for replacement by an amino acid analogue in the polypeptide ofinterest. An amino acid analogue is preferably an analogue of thenatural amino acid to be replaced. To replace a selected natural aminoacid with an amino acid analogue in a polypeptide of interest, anappropriate corresponding aminoacyl-tRNA synthetase must be selected.For example, if an amino acid analogue will replace a methionineresidue, then preferably a methionyl tRNA synthetase is selected.

The host-vector system is grown in media lacking the natural amino acidand supplemented with an amino acid analogue, thereby producing amodified polypeptide that has incorporated at least one amino acidanalogue. This method is superior to existing methods as it improves theefficiency of incorporation of amino acid analogues into polypeptides ofinterest and increases the quantity of modified polypeptides soproduced.

In an embodiment of the invention, where the host-vector system is anauxotrophic system, the host-vector system is initially grown in mediawhich includes all essential amino acids, induced to express thepolypeptide of interest, and subsequently after induction, is grown inmedia lacking the natural amino acid and supplemented with an amino acidanalogue, thereby producing a modified polypeptide that has incorporatedat least one amino acid analogue.

For example, the method of the invention can be practiced by: (1)growing the host-vector system under suitable conditions having thenatural amino acid and under conditions such that the host-vector systemoverexpresses the aminoacyl-tRNA synthetase; (2) collecting and washingcells to remove presence of the natural amino acid; (3) resuspending thecells in media medium which lacks the natural amino acid and has anamino acid analogue; (4) inducing the expression of the polypeptide ofinterest; (5) growing the cells in a medium which lacks the naturalamino acid and has an amino acid analogue under conditions such that thehost-vector system overexpresses the aminoacyl-tRNA synthetase and thepolypeptide molecule of interest; and (6) isolating the modifiedpolypeptide of interest.

In an embodiment of the invention, the polypeptide of interest isdihydrofolate reductase, the natural amino acid is methionine, theaminoacyl-tRNA synthetase is methionyl tRNA synthetase, and the aminoacid analogues of methionine are 6,6,6-trifluoromethionine,homoallylglycine, homoproparglycine, norvaline, norleucine,cis-crotylglycine, trans-crotylglycine, 2-aminoheptanoic acid,2-butynylglycine, allylglycine, azidoalanine and azidohomoalanine.

Polypeptides of Interest

In accordance with the invention, the polypeptides may be from anysource whether natural, synthetic, semi-synthetic, or recombinant. Theseinclude hormones, enzymes and protein fibers. Of these proteins,well-known examples are insulin, interferons, growth hormones, serumalbumin and epidermal growth factor.

The polypeptides of interest can be those which wild-type cells cannotnaturally produce. In view of the diversity of the modified polypeptidesthat can be produced using the methods of the invention, it ispreferable that the polypeptide of interest be different from thoseproduced by wild type cells.

Natural Amino Acids

Natural amino acids are amino acid residues that will be replaced in apolypeptide of interest by a desired amino acid analogue using themethods of the invention.

Amino acids constituting a natural amino acid residue may be selectedfrom the 20 natural amino acids, namely glycine, alanine, valine,leucine, isoleucine, serine, threonine, aspartic acid, glutamic acid,asparagine, glutamine, lysine, arginine, cysteine, methionine,phenylalanine, tyrosine, tryptophan, histidine and proline, thatconstitute the amino acid sequence of a polypeptide of interest.

Aminoacyl-tRNA Synthetases

Aminoacyl-tRNA synthetases can be from any source whether natural,synthetic, semi-synthetic or recombinant (mutated or geneticallyengineered). Accordingly, the aminoacyl-tRNA synthetases can be from anyeukaryotic or prokaryotic cell. Aminoacyl-tRNA synthetases can haveoriginated from the same or different cell as the host cell. Types ofaminoacyl-tRNA synthetases can include but are not limited to glycine,alanine, valine, leucine, isoleucine, serine, threonine, aspartic acid,glutamic acid, asparagine, glutamine, lysine, arginine, cysteine,methionine, phenylalanine, tyrosine, tryptophan, histidine and prolinet-RNA synthetases. In accordance with the invention, selection of anappropriate aminoacyl-tRNA synthetase depends on the natural amino acidso selected to be replaced by an amino acid analogue. For example, if anamino acid analogue will replace methionine, then a methionyl tRNAsynthetase is used.

It may be possible to use genetically engineered aminoacyl-tRNAsynthetases that recognize amino acid analogues and are able tofacilitate the incorporation of that amino acid analogue into apolypeptide. For example, hydroxy acids can be incorporated to form anester linkage in place of an amide linkage of polypeptides.

Aminoacyl-tRNA synthetases can be mutated or genetically engineered toenhance properties of the enzyme to facilitate the incorporation of theamino acid analogues into polypeptides of interest. For example, theediting function of the aminoacyl-tRNA synthetases can be eliminated.

Nucleic acid sequences encoding the appropriate aminoacyl-tRNAsynthetase are used in the methods of the invention.

Amino Acid Analogues

The amino acid analogues incorporated into polypeptides using themethods of this invention are different from the twenty naturallyoccurring counterparts in their side chain functionality. The amino acidanalogue can be a close analogue of one of the twenty natural aminoacids, or it can introduce a completely new functionality and chemistry.The amino acid analogue can replace an existing amino acid in a protein(substitution).

There may be a variety of amino acid analogues that can be added to amedium according to the present invention. Suitable amino acid analoguesinclude, but are not limited to, molecules having fluorinated,electroactive, conjugated, azido, carbonyl, alkyl and unsaturated sidechain functionalities. The following are representative examples ofamino acid analogues:

Amino acid analogues which are modifications of natural amino acids inthe side chain functionality, such that the imino groups or divalentnon-carbon atoms such as oxygen or sulfur of the side chain of thenatural amino acids have been substituted by methylene groups, or,alternatively, amino groups, hydroxyl groups or thiol groups have beensubstituted by methyl groups, olefin, or azido groups, so as toeliminate their ability to form hydrogen bonds, or to enhance theirhydrophobic properties (e.g., methionine to norleucine).

Amino acid analogues which are modifications of natural amino acids inthe side chain functionality, such that the methylene groups of the sidechain of the natural amino acids have been substituted by imino groupsor divalent non-carbon atoms or, alternatively, methyl groups have beensubstituted by amino groups, hydroxyl groups or thiol groups, so as toadd ability to form hydrogen bonds or to reduce their hydrophobicproperties (e.g., leucine to 2-aminoethylcysteine, or isolecine too-methylthreonine).

Amino acid analogues which are modifications of natural amino acids inthe side chain functionality, such that a methylene group or methylgroups have been added to the side chain of the natural amino acids toenhance their hydrophobic properties (e.g., Leucine togamma-Methylleucine, Valine to beta-Methylvaline (t-Leucine)).

Amino acid analogues which are modifications of natural amino acids inthe side chain functionality, such that methylene groups or methylgroups of the side chain of the natural amino acids have been removed toreduce their hydrophobic properties (e.g., Isoleucine to Norvaline).

Amino acid analogues which are modifications of natural amino acids inthe side chain functionality, such that the amino groups, hydroxylgroups or thiol groups of the side chain of the natural amino acids havebeen removed or methylated to eliminate their ability to form hydrogenbonds (e.g., Threonine to o-methylthreonine or Lysine to Norleucine).

Optical isomers of the side chains of natural amino acids (e.g.,Isoleucine to Alloisoleucine);

Amino acid analogues which are modifications of natural amino acids inthe side chain functionality, such that the substituent groups have beenintroduced as side chains to the natural amino acids (e.g., Asparagineto beta-fluoroasparagine).

Amino acid analogues which are modifications of natural amino acidswhere the atoms of aromatic side chains of the natural amino acids havebeen replaced to change the hydrophobic properties, electrical charge,fluorescent spectrum or reactivity (e.g., Phenylalanine toPyridylalanine, Tyrosine to p-Aminophenylalanine).

Amino acid analogues which are modifications of natural amino acidswhere the rings of aromatic side chains of the natural amino acids havebeen expanded or opened so as to change hydrophobic properties,electrical charge, fluorescent spectrum or reactivity (e.g.,Phenylalanine to Naphthylalanine, Phenylalanine to Pyrenylalanine).

Amino acid analogues which are modifications of the natural amino acidsin which the side chains of the natural amino acids have been oxidizedor reduced so as to add or remove double bonds (e.g., Alanine toDehydroalanine, Isoleucine to Beta-methylenenorvaline).

Amino acid analogues which are modifications of proline in which thefive-membered ring of proline has been opened or, additionally,substituent groups have been introduced (e.g., Proline toN-methylalanine).

Amino acid analogues which are modifications of natural amino acids inthe side chain functionality, in which the second substituent group hasbeen introduced at the alpha-position (e.g., Lysine toalpha-difluoromethyllysine).

Amino acid analogues which are combinations of one or more alterations,as described supra (e.g., Tyrosine to p-Methoxy-m-hydroxyphenylalanine).

Amino acid analogues which differ in chemical structures from naturalamino acids but can serve as substrates for aminoacyl-tRNA synthetase byassuming a conformation analogous to natural amino acids when bound tothis enzyme (e.g., Isoleucine to Furanomycin).

Types of amino acid analogues of methionine are6,6,6-trifluoromethionine, homoallylglycine, homoproparglycine,norvaline, norleucine, cis-crotylglycine, trans-crotylglycine,2-aminoheptanoic acid, 2-butynylglycine, allylglycine, azidoalanine andazidohomoalanine.

Vectors

In accordance with the methods of the invention, suitable expressionvectors which may be used include, but are not limited to, viralparticles, baculovirus, phage, plasmids, phagemids, cosmids, phosmids,bacterial artificial chromosomes, viral DNA (e.g., vaccinia, adenovirus,foul pox virus, pseudorabies and derivatives of SV40), P1-basedartificial chromosomes, yeast plasmids, yeast artificial chromosomes,and any other vectors specific for specific hosts of interest (such asbacillus, aspergillus, yeast, etc.). Such vectors can be chromosomal,nonchromosomal or synthetic DNA sequences.

Large numbers of suitable vectors are known to those of skill in theart, and are commercially available. The following vectors are providedby way of example; Bacterial: pQE70, pQE60, pQE-9, pQE15 (Qiagen,Valencia, Calif.), psiX174, pBluescript SK, pBluescript KS, (Stratagene,La Jolla, Calif.); pTRC99a, pKK223-3, pKK233-3, pDR540, pRIT2T(Pharmacia, Uppsala, Sweden); Eukaryotic: pWLNEO, pXT1, pSG (Stratagene,La Jolla, Calif.) pSVK3, pBPV, PMSG, pSVLSV40 (Pharmacia, Uppsala,Sweden).

A preferred vector for expression may be an autonomously replicatingvector comprising a replicon that directs the replication of the nucleicacids within the appropriate host cell. The preferred vectors alsoinclude an expression control element, such as a promoter sequence,which enables transcription of the inserted sequences and can be usedfor regulating the expression (e.g., transcription and/or translation)of an operably linked sequence in an appropriate host cell such asEscherichia coli. Methods for generating vectors are well known in theart, for example, see Maniatis, T., et al., 1989 Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y., incorporated by reference herein.

Expression control elements are known in the art and include, but arenot limited to, inducible promoters, constitutive promoters, secretionsignals, enhancers, transcription terminators, and other transcriptionalregulatory elements. Other expression control elements that are involvedin translation are known in the art, and include the Shine-Dalgarnosequence, and initiation and termination codons. The preferred vectoralso includes at least one selectable marker gene that encodes a geneproduct that confers drug resistance, such as resistance to ampicillinor tetracycline. The vector also comprises multiple endonucleaserestriction sites that enable convenient insertion of exogenous DNAsequences.

The preferred vectors for generating polypeptides of interest are thosecompatible to prokaryotic host cells. Prokaryotic cell expressionvectors are well known in the art and are available from severalcommercial sources. For example, a pQE vector (e.g., pQE15, availablefrom Qiagen Corp., Valencia, Calif.) may be used to express polypeptidesof interest, containing natural amino acids and modified polypeptides,including those containing amino acid analogues, in bacterial hostcells.

The nucleic acids derived from a microorganism(s) may be inserted intothe vector by a variety of procedures. In general, the nucleic acids canbe inserted into an appropriate restriction endonuclease site(s) byprocedures known in the art. Such procedures and others are deemed to bewithin the scope of those skilled in the art.

The nucleic acid sequence encoding the aminoacyl-tRNA synthetase orpolypeptide of interest in the expression vector may be operativelylinked to an appropriate expression control sequence(s) (promoter) todirect mRNA synthesis. Bacterial promoters include lacI, lacZ, T3, T7,gpt, lambda P_(R), P_(L) and trp. Eukaryotic promoters include CMVimmediate early, HSV thymidine kinase, early and late SV40, LTRs fromretrovirus, and mouse metallothionein-I.

Selection of the appropriate vector and its correlative promoter is wellwithin the level of ordinary skill in the art. The expression vector mayalso contain a ribosome binding site for translation initiation and atranscription terminator. The vector may also include appropriatesequences for amplifying expression. Promoter regions can be selectedfrom any desired gene using CAT (chloramphenicol transferase) vectors orother vectors with selectable markers. Chemical or temperature sensitivepromotors can be used for inducing the expression of either theaminoacyl-tRNA synthetase or the target protein.

In addition, the expression vectors preferably contain one or moreselectable marker genes to provide a phenotypic trait for selection oftransformed host cells, such as dihydrofolate reductase or neomycinresistance for eukaryotic cell culture, or tetracycline or ampicillinresistance in E. coli.

Inducers

In accordance with the methods of the invention when the expressioncontrol element is an inducible promotor, the promoter may be induced byan external stimulus, such as by adding a compound (e.g., IPTG) or byheat shocking to initiate the expression of the gene.

Level of Expression

In accordance with the methods of the invention, the production of theaminoacyl-tRNA synthetase and/or the polypeptide of interest ispreferably in an amount in excess of the level (any increase that ismeaningful or confers a benefit) produced by a naturally occurring geneencoding the aminoacyl-tRNA synthetase and/or the polypeptide ofinterest.

The increase in the level of aminoacyl-tRNA synthetase and/or thepolypeptide of interest can be measured by monitoring an increase inprotein expression by gel electrophoresis, western blot analysis, orother relevant methods of protein detection (Maniatis, T., et al., 1989Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.).

The increase in the level of aminoacyl-tRNA synthetase can also bedetermined by measuring the ATP-PP_(i) exchange activity (Mellot, P.;Mechulam, Y.; LeCorre, D.; Blanquet, S.; Fayat, G. J. Mol. Biol. 1989,208, 429; Blanquet, S.; Fayat, G.; Waller, J.-P. Eur. J. Biochem. 1974,44, 343; Ghosh, G.; Pelka, H.; Schulman, L. H. Biochemistry 1990, 29,2220) of cell lysates.

Fusion Genes

In accordance with the methods of the invention, a fusion gene includesa sequence encoding a polypeptide of the invention operatively fused(e.g., linked) to a non-related sequence such as, for example, a tagsequence to facilitate isolation and/or purification of the expressedgene product (Kroll, D. J., et al., 1993 DNA Cell Biol 12:441-53). ThepQE expression vectors used in this invention express proteins fused toa poly-Histidine tag that facilitates isolation and/or purification ofthe expressed gene.

Host Cells

In accordance with the methods of the invention, types of host cellsinclude, but are not limited to, bacterial cells, such as E. coli,Streptomyces, Salmonella typhimurium; fungal cells, such as yeast;insect cells such as Drosophila S2 and Spodoptera Sf9; animal cells suchas CHO, COS or Bowes melanoma; adenoviruses; plant cells, etc. Theselection of an appropriate host is deemed to be within the scope ofthose skilled in the art from the teachings herein.

A preferred embodiment of a host cell is an auxotroph. Auxotrophs dependupon the external environment to supply certain amino acids, forexample, a methionine auxotroph depends on methionine in the growthmedium for its survival. The choice of auxotroph is dependent on theamino acid that is selected to be replaced by an amino acid analogue inthe target protein (e.g., if methionine is selected, then a methionineauxotroph is employed, if phenylalanine is selected, then aphenylalanine auxotroph is employed).

Suitable auxotrophs include, but are not limited to CAG18491, B834(DE3),AD494, DL41, and ML304d.

Host cells may be either wild type cells or transformants. The term“transformants” as used herein including products of transformation,transfection and transduction. Preferably, the polypeptides of interestto be produced by the cells according to the present invention are thosewhich wild-type cells cannot produce. Thus, it is preferable that thecells to be used in the present invention be transformants.

Host-Vector Systems

The invention further discloses a host-vector system comprising a vectoror vectors having nucleic acids encoding the aminoacyl-tRNA synthetaseand polypeptide of interest.

The host-vector system is used to produce the polypeptides of interest.The host cell can be either prokaryotic or eukaryotic. Examples ofsuitable prokaryotic host cells include bacterial strains from generasuch as Escherichia, Bacillus, Pseudomonas, Streptococcus, andStreptomyces. Examples of suitable eukaryotic host cells include a yeastcell, a plant cell, or an animal cell, such as a mammalian cell.

Introduction of the vectors of the present invention into an appropriatecell host is accomplished by well known methods that typically depend onthe type of vector used and host system employed. For transformation ofprokaryotic host cells, electroporation and salt treatment methods aretypically employed, see for example, Cohen et al., 1972 Proc Acad SciUSA 69:2110; Maniatis, T., et al., 1989 Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.Transformation of vertebrate cells with vectors by electroporation,cationic lipid or salt treatment methods, is typically employed, see,for example, Graham et al, 1973 Virol 52:456; Wigler et al., 1979 ProcNatl Acad Sci USA 76:1373-76.

Successfully transformed host cells, i.e., cells that contain a vectorof the present invention, are identified by well-known techniques. Forexample, cells resulting from the introduction of a vector of thepresent invention are selected and cloned to produce single colonies.Cells from those colonies are harvested, lysed and their nucleic acidcontent examined for the presence of the vector using a method such asthat described by Southern, J Mol Biol (1975) 98:503, or Berent et al.,Biotech (1985) 3:208, or the proteins produced from the cell are assayedvia a biochemical assay or immunological method such as Westernblotting.

The methods of the invention in which the cloned gene is expressed in asuitable host cell are preferred if longer polypeptides, higher yield,or a controlled degree of amino acid analogue incorporation is desired.For example, a suitable host cell is introduced with an expressionvector having the nucleotide sequence encoding the polypeptide ofinterest. The host cell is then cultured under conditions that permit invivo production of the desired polypeptide, wherein one or morenaturally occurring amino acids in the desired polypeptide are replacedwith the amino acid analogues and derivatives.

A preferred embodiment provides a host-vector system comprising thepQE15 (Qiagen, Santa Clara, Calif.) vector having a sequence encodingthe aminoacyl-tRNA synthetase and target polypeptide of the invention,which is introduced along with the pREP4 (Qiagen) vector into anappropriate auxotrophic host cell such as E. coli methionine auxotrophCAG18491 strain, which is useful, for example, for producing apolypeptide where a selected natural amino acid is replaced with anamino acid analogue.

An embodiment of the host cells of the present invention are Escherichiacoli and transformants thereof, and an example of the protein to beproduced is dihydrofolate reductase.

Media

Suitable media for growing the host-vector systems of the invention arewell known in the art, for example, see Sambrook et al., MolecularCloning (1989), supra. In general, a suitable media contains all theessential nutrients for the growth of the host-vector system. The mediacan be supplemented with antibiotics that are selected for host-vectorsystem.

The media may contain all 20 natural amino acids or lack a selectednatural amino acid. The media may also contain an amino acid analogue inplace of a selected natural amino acid.

Potential Uses of Modified Polypeptides

According to the present invention, it is now possible to createentirely new modified polypeptides, in which amino acid analogues, aswell as the 20 natural amino acids are used as constituents.

Modified polypeptides can be used to prepare functional drugs,antagonistic drugs or inhibitory agents. Also, using non-natural aminoacids in protein engineering expands the potential designs ofpolypeptides. Since such modified polypeptides are not natural, they maybe less susceptible to proteolytic enzymes generally present in cells.

Introduction of amino acid analogues in polypeptides may producemodified polypeptides having a variety of side chains having highlyactive chemical functional groups. The reactivity of the various typesof the functional groups introduced can be exploited to control proteinstructure and function. For example, polypeptides or proteins may beproduced that have undergone site-specific phosphorylation, methylationor addition of sugar chains. It may be possible to produce modifiedpolypeptides as derivatives analogous to specified proteins by theintroduction of amino acid analogues having functional groups to formcrosslinks so that cellular components which interact with the specifiedproteins in the cells can be detected. Modified polypeptides withincorporated fluorescent amino acid residues are useful to tracemetabolic pathways in organisms or to elucidate mechanisms of biologicalactions. It is possible to produce modified polypeptides having aminoacid analogues which differ in acid dissociation constant from naturalamino acids, so as to control properties of the polypeptides that dependon the acidity in aqueous solutions.

It is possible to introduce amino acid analogues into polypeptides thatwill self-assemble so as to mimic viruses (e.g., coat proteins), musclefibers (e.g., actin and myosin) or chromatin (e.g., histones) so as tocreate supra-molecular structures having specified functions.Additionally, the supra-molecular structures can be further modified ina biological system to create other supra-molecular structures havingspecified functions.

It may be possible to add amino acid analogues according to the methodsof the invention to artificial feeds for silk worms that can synthesizesilk with the amino acid analogues. Further, it may be possible toproduce protein fibers with optical properties from modifiedpolypeptides into which amino acid analogues have been incorporated. Inthis regard, modified polypeptides with amino acid analogues havingfunctional groups to form crosslinkages can produce supra-molecularstructures with silk as supporting construction. Crosslinkages of themodified polypeptides can then produce new proteinaceous structures.Into the structures thus produced, non-natural fluorescent amino acidscan be introduced, e.g., to make biochips for photoenergy transduction.

ADVANTAGES OF THE INVENTION

The invention introduces a unique strategy that can be widely applied toincorporate amino acid analogues to substitute for any of the selectednatural amino acid residues in polypeptides of interest. A greater rangeof amino acid analogues can be employed for protein synthesis. Inaddition, modified polypeptides produced using the methods of thisinvention can be produced in higher yields and with high levels ofreplacement of natural amino acids.

The method of this invention changes the building blocks of proteinsynthesis, leaving the “blueprint” encoding the proteins unchanged. Theinvention, therefore, permits a rapid and predictable approach toprotein design and produces modified polypeptides with significantlyincreased yields and expansion of amino acid analogues that can serve assubstrates for polypeptide synthesis.

This method of this invention is generally applicable to a large rangeof proteins, enzymes, and peptides, and is not limited by the size orstructure of the proteins or polypeptides. Incorporation of amino acidanalogues with different functionalities, such as double bonds, can beutilized for further chemical derivatization of the polypeptide ofinterest. Furthermore, the feasibility of incorporating amino acidanalogues using in vivo methods should allow the manipulation ofenzymes, signaling molecules, protein ligands, and may prove to be ofbroad utility in the engineering of more versatile biologicalassemblies.

The following examples are presented to illustrate the present inventionand to assist one of ordinary skill in making and using the same. Theexamples are not intended in any way to otherwise limit the scope of theinvention.

EXAMPLE I

This example demonstrates the selectivity of methionyl t-RNA synthetasefor methionine analogues and the efficient incorporation of unsaturatedmethionine analogues into proteins in vivo.

Synthesis of Amino Acid Analogues

Each of the analogues 2-7 and 11 (FIG. 2) was prepared by alkylation ofdiethyl acetamidomalonate with the appropriate alkyl tosylate followedby decarboxylation and deprotection of the amine function. This sectionprovides information on general synthetic procedures and a detailedprotocol for preparation of 2. Similar methods were used to prepare 3-7and 11. Analogues 8, 9, 12, and 13 are available commercially(Sigma-Aldrich, St. Louis, Mo.). Analogue 10 was prepared as describedby Blackwell et al. (H. E. Blackwell, R. H. Grubbs, Angew. Chem. 1998,110, 3469-3472; Angew. Chem. Int. Ed. 1998, 37, 3281-3284.).

General Procedures.

Glassware was dried at 150° C. and cooled under nitrogen prior to use.Tetrahydrofuran (THF) was freshly distilled from Na/benzophenone.N,N-Dimethylformamide (DMF) was distilled and stored over BaO. Pyridine(99.8%, anhydrous, Aldrich) and other reagents and solvents were used asreceived. ¹H NMR spectra were recorded on Bruker AC 200 and AMX 500spectrometers and ¹³C NMR spectra were recorded on a Bruker DPX 300spectrometer. Column chromatography was performed with silica gel 60,230-400 mesh (EM Science); silica 60-F254 (Riedel-de Haen) was used forthin layer chromatography.

DL-2-amino-5-hexenoic Acid (2)

(Drinkwater, D. J.; Smith, P:W. G. J. Chem. Soc. C 1971, 1305; Baldwin,J. E.; Hulme, C.; Schofield, C. J. J. Chem. Res. (S) 1992, 173).

3-Buten-1-ol 4-methylbenzene Sulfonate.

A solution of 3 g (42 mmol) 3-buten-1-ol in 10 mL dry pyridine wascooled in an ice bath. Tosyl chloride (7.9 g, 42 mmol), was added. Afterstirring for 3 h the mixture was poured into 30 mL of anice/concentrated HCl 4/1 v/v solution, extracted with 60 mL diethylether and dried overnight in the freezer over MgSO₄. The mixture wasfiltered and the ether evaporated to yield 7.22 g (76%) of 3-buten-1-ol4-methylbenzene sulfonate as a yellow oil. ¹H NMR (CDCl₃): δ 2.39-2.53(m, 2H, J=6.5 and 6.9 Hz, CH ₂—CH═CH₂; and s, 3H, CH ₃—Ar), 4.08 (t,J=6.5 Hz, 2H, CH ₂OSO₂), 5.09-5.15 (m, 2H, J_(Z)=10.4, J_(E)=16.6,J_(gem)=3.1 Hz, CH₂—CH═CH ₂), 5.57-5.82 (m, 1H, J_(Z)=10.4, J_(E)=16.6,J=6.9 Hz, CH₂—CH═CH₂), 7.38 and 7.72 (d, 4H, J=8.6 Hz, Ar).

Acetylamino-3-butenyl Propanedioic Acid Diethyl Ester.

Diethyl acetamidomalonate, 1.56 g (6.9 mmol), was dissolved at roomtemperature under N₂ in 10 mL dry THF. Potassium tert-butoxide (0.80 g,7 mmol), was added under vigorous stirring. The mixture was heated for 2h at 60° C. 3-Buten-1-ol 4-methylbenzenesulfonate (1.5 g, 6.9 mmol) wasadded, and the mixture was heated under reflux for 2 days. The THF wasremoved, the residue was quenched with 10 mL 1 M HCl, and the crudeproduct was extracted with ethyl acetate (25 mL). The ethyl acetatesolution was washed twice with 25 mL water, dried over MgSO₄, filteredand concentrated. The crude product was purified by columnchromatography (eluent cyclohexane/ethyl acetate 2/1 v/v) to yield 0.82g (44%) of acetylamino-3-butenyl-propanedioic acid diethyl ester. ¹H NMR(CDCl₃): δ 1.28 (t, 6H, J=7.2 Hz, CH ₃—CH₂), 1.78-2.0 (m, 2H, J=8.3, 6.5Hz, CH₂═CH—CH ₂—CH₂), 2.08 (s, 3H, CONH—CH ₃), 2.45 (m, 2H, J=8.3 Hz,CH₂═CH—CH₂—CH ₂), 4.25 (q, 4H, J=7.2 Hz, CH₃—CH ₂), 4.90-5.09 (m, 2H,J_(Z)=10.4, J_(E)=16.6, J_(gem)=3.2 Hz, CH₂—CH═CH ₂), 5.61-5.90 (m, 1H,J_(Z)=10.4, J_(E)=16.6, J=6.5 Hz, CH₂—CH═CH₂), 6.78 (s, 1H, CONH—CH₃).

DL-2-amino-5-hexenoic Acid.

The diethyl ester obtained as described above was hydrolyzed to thedicarboxylate by heating under reflux for 4 h in 25 mL 10 wt % NaOH. Thesolution was neutralized with 6 M HCl and the solvent was evaporated.The diacid was extracted with 25 mL of methanol. Following solventevaporation, 20 mL 1M HCl was added and the solution was refluxed for 3h. The solvent was evaporated and the product was taken up in 10 mLmethanol. Propylene oxide (5 mL) was added and the mixture was stirredovernight at room temperature. The precipitate was filtered and dried,yielding DL-2-amino-5-hexenoic acid (0.47 g, 63%). The product wasrecrystallized from EtOH/H₂O 2/1 v/v (0.28 g, 60%). The ¹H NMR data werein agreement with those of reference 16 (Hatanaka, S. I.; Furukawa, J.;Aoki, T.; Akatsuka, H.; Nagasawa, E. Mycoscience, 1994, 35, 391). ¹H NMR(D₂O): δ 1.78-2.0 (m, 2H, J=6.4, 6.6 Hz, CH₂═CH—CH ₂—CH₂), 2.08-2.20 (m,2H, J=6.1, 6.4 Hz, CH₂═CH—CH₂—CH ₂), 3.75 (t, 1H, J=6.1 Hz,H₂N—CH—COOH), 4.90-5.12 (m, 2H, J_(Z)=10.5, J_(E)=16.7, J_(gem)=3.3 Hz,CH₂—CH═CH ₂), 5.61-5.90 (m, 1H, J_(Z)=10.5, J_(E)=16.7, J=6.6 Hz,CH₂—CH═CH₂). ¹³C NMR (D₂O): δ 28.9 (CH₂═CH—CH₂—CH₂), 29.9(CH₂═CH—CH₂—CH₂), 54.4 (H₂N—CH—COOH), 116.3 (CH₂—CH═CH₂), 137.3(CH₂—CH═CH₂), 174.8 (COOH).

Determination of Translational Activity

Buffers and media were prepared according to standard protocols(Sambrook, J.; Fritsch, E. F.; Maniatis, T. Molecular Cloning: ALaboratory Manual, 2nd Ed. Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. 1989; Ausubel, F. M.; Brent, K.; Kingston, K. E.;Moore, D. D.; Scidman, J. G.; Smith, J. A.; Struhl, K. Current Protocolsin Molecular Biology, John Wiley & Sons, NY 1995). The E. colimethionine auxotroph CAG18491 (λ⁻, rph-1, metE3079.:Tn10) (obtained fromthe Yale E. coli Genetic Stock Center), was transformed with plasmidspREP4 and pQE15 (Qiagen, Valencia, Calif.), to obtain the expressionhost CAG18491/pQE15/pREP4.

Protein Expression (5 mL Scale).

M9AA medium (50 mL) supplemented with 1 mM MgSO₄, 0.2 wt % glucose, 1mg/L thiamine chloride and the antibiotics ampicillin (200 mg/L) andkanamycin (25 mg/L) was inoculated with 2 mL of an overnight culture ofCAG18491/pQE15/pREP4. When the turbidity of the culture reached anoptical density at 600 nm (OD₆₀₀) of 0.8, a medium shift was performed.The cells were sedimented for 10 min at 3030 g at 4° C., the supernatantwas removed, and the cell pellet was washed twice with 20 mL of M9medium. Cells were resuspended in 50 mL of the M9AA medium describedabove, without methionine. Test tubes containing 5 mL aliquots of theresulting culture were prepared, and were supplemented with 200 μL 1mg/mL (0.27 mM) L-methionine (1) (positive control),DL-2-amino-5-hexenoic acid (2) (0.31 mM), DL-homopropargylglycine (3)(0.31 mM), cis- or trans-DL-2-amino-4-hexenoic acid (4 or 5) (0.31 mM),DL-6,6,6-trifluoro-2-amino hexanoic acid (6) (0.22 mM),DL-2-aminoheptanoic acid (7) (0.28 mM), L-norvaline (8) (0.34 mM) orL-norleucine (9). (0.31 mM), respectively. A culture lacking methionine(or any analogue) served as the negative control. Protein expression wasinduced by addition of isopropyl-β-D-thiogalactopyranoside (IPTG) to afinal concentration of 0.4 mM. Samples were taken every hour for 4 h,the OD₆₀₀ was measured, and the samples were sedimented. After thesupernatant was decanted, the cell pellets were resuspended, in 20 μLdistilled H₂O.

Protein expression was monitored by SDS polyacrylamide gelelectrophoresis (12% acrylamide running gel, 12 mA, 14 h), using anormalized OD₆₀₀ of 0.2 per sample.

Protein Expression (1 L Scale)

Similar procedures were used for preparation and isolation of mDHFR frommedia supplemented with 1, 2 or 3. The example presented is for mediumsupplemented with 3. M9AA medium (100 mL) supplemented with 1 mM MgSO₄0.2 wt % glucose, 1 mg/L thiamine chloride and the antibioticsampicillin (200 mg/L) and kanamycin (25 mg/L) was inoculated with E.coli strain CAG18491/pQE15/pREP4 and grown overnight at 37° C. Thisculture was used to inoculate 900 mL M9AA medium supplemented asdescribed. The cells were grown to an OD₆₀₀ of 0.94 and the medium shiftwas performed as described for the small scale experiments, followed byaddition of 40 mL of 1 mg/mL DL-homopropargylglycine (3). IPTG (0.4 mM)was added, and samples were taken at 1 hour intervals. OD₆₀₀ wasmeasured, the samples were sedimented and decanted, and the cell pelletswere resuspended in 20 μL distilled H₂O. Protein expression wasmonitored by SDS polyacrylamide gel electrophoresis (12% acrylamiderunning gel, 12 mA, 15 h).

Protein Purification

Approximately 4.5 h after induction, cells were sedimented (9,800 g, 10min, 4° C.) and the supernatant was removed. The pellet was placed inthe freezer overnight. The cells were thawed for 30 min at 37° C., 30 mLof buffer (6 M guanidine-HCl, 0.1 M NaH₂PO₄, 0.01 M Tris, pH 8) wasadded and the mixture was shaken at room temperature for 1 h. The celldebris was sedimented (15,300 g, 20 min, 4° C.) and the supernatant wassubjected to immobilized metal affinity chromatography (Ni-NTA resin)according to the procedure described by Qiagen (The QiagenExpressionist, Purification Procedure 7, 1992, 45). The supernatant wasloaded on 10 mL of resin which was then washed with 50 mL of guanidinebuffer followed by 25 mL of urea buffer (8 M urea, 0.1 M NaH₂PO₄ and0.01 M Tris, pH 8). Similar urea buffers were used for three successive25 mL washes at pH values of 6.3, 5.9 and 4.5, respectively. Targetprotein was obtained in washes at pH 5.9 and 4.5. These washes werecombined and dialyzed (Spectra/Por membrane 1, MWCO=6-8 kDa) againstrunning distilled water for 4 days, followed by batchwise dialysisagainst doubly distilled water for one day. The dialysate waslyophilized to yield 70 mg of modified mDHFR (mDHFR-Y). A similarprocedure using medium supplemented with 2 yielded 8 mg of mDHFR-E. Acontrol experiment in 2×YT medium afforded 60 mg of mDHFR. Amino acidanalyses, electrospray mass spectrometry and N-terminal proteinsequencing was performed on the mDHFR isolated.

Enzyme Purification and Activation Assays

The fully active, truncated form of methionyl tRNA synthetase (MetRS)was purified from overnight cultures of JM101 cells carrying the plasmidpGG3. (The plasmid, which encodes the tryptic fragment of MetRS, Ghosh,G.; Brunie, S.; Schulman, L. H. J. Biol. Chem. 1991, 266, 17136-17141).The enzyme was purified by size exclusion chromotography as previouslydescribed (Mellot, P.; Mechulam, Y.; LeCorre, D.; Blanquet, S.; Fayat,G. J. Mol. Biol. 1989, 208, 429). Activation of methionine analogues byMetRS was assayed via the amino-acid-dependent ATP-PP_(i) exchangereaction, also as previously described (Mellot, P.; Mechulam, Y.;LeCorre, D.; Blanquet, S.; Fayat, G. J. Mol. Biol. 1989, 208, 429;Blanquet, S.; Fayat, G.; Waller, J.-P. Eur. J. Biochem. 1974, 44, 343;Ghosh, G.; Pelka, H.; Schulman, L. H. Biochemistry 1990, 29, 2220). Theassay, which measures the ³²P-radiolabeled ATP formed by theenzyme-catalyzed exchange of ³²P-pyrophosphate (PP_(i)) into ATP, wasconducted in 150 μl of reaction buffer (pH 7.6, 20 mM imidazole; 0.1 mMEDTA, 10 mM β-mercaptoethanol, 7 mM MgCl₂, 2 mM ATP, 0.1 mg/ml BSA, and2 mM PP_(i) (in the form of sodium pyrophosphate with a specificactivity of approximately 0.18 TBq/mole)). Assays to determine if themethionine analogues 2-13 are recognized by MetRS were conducted insolutions 75 nM in enzyme and 5 mM in the L-isomer of the analogue witha reaction time of 20 minutes. Kinetic parameters for analogue 5 wereobtained with an enzyme concentration of 75 nM and analogueconcentrations of 100 μM to 10 mM. Parameters for methionine wereobtained by using concentrations ranging from 10 μM to 1 mM. K_(m)values for methionine matched those previously reported (Ghosh, G.;Pelka, H.; Schulman, L. H.; Brunie, S. Biochemistry 1991, 30, 9569),though the measured k_(cat) was somewhat lower than the literaturevalue. Aliquots of 20 μl were removed from the reaction mixture atvarious time points and were quenched in 0.5 ml of a solution comprising200 mM PP_(i), 7% w/v HClO₄, and 3% w/v activated charcoal. The charcoalwas rinsed twice with 0.5 mL of a 10 mM PP_(i), 0.5% HClO₄ solution andwas then resuspended in 0.5 mL of this solution and counted via liquidscintillation methods. Kinetic constants were calculated by nonlinearregression analysis.

Computation

Single-point energy ab initio calculations (Hartree-Fock model, 6-31G*basis set) (Hehre, W. J.; Ditchfield, R.; Pople, J. A. J. Chem. Phys.1972, 56, 2257; Hariharan, P. C.; Pople, J. A. Chem. Phys. Lett. 1972,66, 217; Francl, M. M. et al. J. Chem. Phys. 1982, 77, 3654) wereperformed for methionine and for analogues 2, 3 and 5 with fullyextended side chains. Electron density maps are shown as surfaces ofelectron density 0.08 electrons/au³. Isopotential plots are representedas surfaces where the energy of interaction between the amino acid and apoint positive charge is equal to −10 kcal/mole. Calculations wereperformed by using the program MacSpartan (Wavefunction, Inc., Irvine,Calif., USA).

Results and Discussion

Methionine Analogues

Methionine analogues 2-13 were investigated with respect to theircapacity to support protein synthesis in E. coli cells depleted ofmethionine. Norvaline (8) and norleucine (9), allylgycine (12), andpropargylglycine (13) are commercially available. Analogues 2-7 and 11were prepared by alkylation of diethyl acetamidomalonate with thecorresponding tosylates via standard procedures, and the remaininganalogues were prepared as described supra. In the cases of the cis- andtrans-crotylglycines (4 and 5) the tosylates were prepared in situ, andbecause of fast exchange of the tosyl group with chloride ion, mixturesof the chloride and the tosylate were obtained. Hydrolysis of themalonate and conversion to the amino acid had to be performed under mildacidic conditions for analogues 2, 4 and 5; treatment with 6 N HCl, orreflux in 1 N HCl for more than 5 h led to HCl addition to the doublebond. In all cases the analogues were obtained as racemates and wereused as such.

Protein Expression

E. coli strain CAG18491/pQE15/pREP4, which produces the test proteinmDHFR upon induction with IPTG, was used as the expression host. Theparent strain CAG18491 is dependent on methionine for growth, owing toinsertion of transposon Tn10 into the metE gene, which is essential forthe final step in the endogenous synthesis of methionine. Cultures weregrown in minimal medium supplemented with methionine until a celldensity corresponding to OD₆₀₀ 0.8-1.0 was reached. Cells weresedimented, washed and resuspended in minimal medium without methionine.Aliquots of the culture were then supplemented with one of the analogues2-13. Protein synthesis was induced with IPTG and cell growth andprotein expression were followed over a 4 h period. Expression resultsare presented in FIG. 3, and show clearly that analogues 2 and 3 exhibittranslational activity sufficient to allow protein synthesis in theabsence of methionine. Analogues 4-8 and 12-13 are not active in theassay reported here, while the known translational activity ofnorleucine (9) was confirmed. CAG18491/pQE15/pREP4 cultures did not growin minimal media in which methionine was replaced by 2 and 3, at thetime of inoculation.

Analysis of Protein Structure

The extent of replacement of methionine by analogues 2 and 3 wasdetermined by an amino acid analysis, N-terminal sequencing, and (for 2)¹H nuclear magnetic resonance spectroscopy (Table 1). Proteinscontaining 2 and 3 were designated mDHFR-E (alkene) and mDHFR-Y(alkyne), respectively. TABLE 1 PROTEIN YIELD AND EXTENT OF METHIONINEREPLACEMENT Replacement (%) Yield Amino Acid Protein (mg)^(a) AnalysisSequencing ¹H NMR mDHFR-E 8 86 92 77 mDHFR-Y 70 100 88 Not determined^(a)Yield of purified protein obtained from 1 L of CAG18491/pQE15/pREP4culture grown to OD₆₀₀ = 0.94 prior to induction by addition of IPTG.The yield of mDHFR obtained from control cultures supplemented withmethionine was approximately 70 mg/L.

mDHFR-E.

Amino acid analysis of mDHFR-E showed a methionine content of 0.5 mol %vs. the value of 3.8 mol % expected for mDHFR. Although 2 appears to beunstable under the conditions used to hydrolyze the protein for aminoacid analysis, assumption that the decrement in methionine content isdue to replacement by 2 affords an estimate of 86% substitution by theanalogue. This estimate is consistent with the results of N-terminalsequencing of mDHFR-E (FIG. 4), which indicates 92% occupancy of theinitiator site by 2. In the chromatograms shown in FIG. 4, the signaldue to methionine appears at a retention time of 12.3 min, while thatfrom 2 elutes at 14.3 min. The retention time of the signal arising from2 was verified by analysis of an authentic sample of the analogue.Retention of the N-terminal residue in mDHFR was expected on the basisof the known correlation between the extent of methionine excision fromE. coli proteins and the identity of the penultimate amino acid residue(Hirel, P. H.; Schmitter, J. M.; Dessen, P.; Fayat, G.; Blanquet, S.Proc. Natl. Acad. Sci. USA 1989, 86, 8247). Finally, direct evidence forincorporation of the alkene function of 2 was obtained from ¹H NMRspectroscopy. The vinyl CH resonance of 2 appears at a chemical shift of5.7 ppm in the spectrum of mDHFR-E, and can be integrated to yield anestimate of 77% replacement of methionine by the unsaturated analogue. Ayield of 8 mg of mDHFR-E was obtained from a 1 L culture ofCAG18491/pQE15/pREP4 grown in M9AA medium supplemented with 2, comparedwith 70 mg obtained from a similar experiment in medium supplementedwith methionine.

mDHFR-Y.

Methionine could not be detected via amino acid analysis of mDHFR-Y,suggesting quantitative replacement of methionine by the alkyne analogue3. N-terminal sequencing (FIG. 4) indicated 88% occupancy of theinitiator site by 3. ¹H NMR analysis of mDHFR-Y was consistent withnear-quantitative replacement of methionine, as the thiomethyl resonanceat 2.05 ppm—which is prominent in the spectrum of mDHFR—could not bedetected. New signals at 2.2-2.3 ppm—which are not observed in thespectrum of mDHFR and which correspond to signals due to the β- andε-protons of 3—appeared in the spectrum of mDHFR-Y, but were notintegrated carefully owing to overlap with neighboring resonances. Theyield of mDHFR-Y obtained from M9AA medium supplemented with 3 wasessentially identical to that of mDHFR isolated from media supplementedwith methionine.

Enzyme Assays

The relative rates of activation of methionine and methionine analogues2-13 by MetRS were estimated by the ATP-PP_(i) exchange assay. Theresults shown in FIG. 5 illustrate the amount of PP_(i) exchanged at areaction time of 20 minutes under standard assay conditions (seeExperimental Section). Methionine (1) is activated most efficiently bythe enzyme, causing exchange of 9 mmoles of PP_(i) over the time courseof the reaction. Analogues 2 and 3 cause exchange of PP_(i) at ratessimilar to that of norleucine (9), while the remaining analogues 4, 6-8,and 12-13 cause exchange of PP_(i) at levels no higher than background(FIG. 5, lane 14). The background (lane 14) is given for a reactionmixture lacking both the enzyme and the amino acid. Although analogues 5and 11 effect very slow exchange of PP_(i), the activation rate isapparently too low to support protein synthesis at a level that isdetectable in the in vivo assays. Kinetic parameters were determined formethionine and 5 as outlined in the Experimental Section. Comparison ofthe k_(cat)/K_(m) values obtained for methionine (0.54 s⁻¹μM⁻¹) and 5(1.1×10⁻⁴ s⁻¹μM⁻¹) show that 5 is activated 4700-fold less efficientlythan methionine by MetRS. Comparison of the k_(cat)/K_(m) valuesobtained for methionine (0.54 s⁻¹μM⁻¹) and 11 (3.9×10⁻⁵ s⁻¹μM⁻¹) showthat 11 is activated 13825-fold less efficiently than methionine byMetRS.

Discussion

A bacterial host strain (designated CAG18491/pQE15/pREP4) suitable fortesting the translational activity of methionine analogues 2-8 and 10-13was prepared by transformation of E. coli strain CAG18491, a methionineauxotroph, with the repressor plasmid pREP4 and the expression plasmidpQE15. pQE15 encodes mouse dihydrofolate reductase (mDHFR) under controlof a bacteriophage T5 promoter, and appends to mDHFR an N-terminalhexahistidine sequence that facilitates purification of the protein byimmobilized metal affinity chromatography. mDHFR contains eightmethionine residues, each a potential site for substitution by analogues2-8 and 10-13. The translational activity of each analogue was assayedon the basis of its capacity to support synthesis of mDHFR in culturesof CAG18491/pQE15/pREP4 that had been depleted of methionine. In thoseinstances in which the test protein was detected by gel electrophoresis(i.e., for 2 and 3), the modified mDHFR was purified and analyzed todetermine the extent of methionine replacement by the analogue.

The results of the in vivo assays illustrated in FIG. 3 show clearlythat homoallylglycine (2) and homopropargylglycine (3) serve effectivelyas methionine surrogates in bacterial protein synthesis. In contrast,analogues 4-8 and 10-13 do not support measurable levels of proteinsynthesis in bacterial cultures depleted of methionine. It is highlyunlikely that recognition by the elongation factors of the ribosome ortransport into the cell are the limiting factors for incorporation ofthese analogues. The ribosome is remarkably permissive toward amino acidanalogues with widely varying chemical functionality, as has beendemonstrated by the numerous analogues incorporated into proteins in invitro translation experiments (Cornish, V. W.; Mendel, D.; Schultz, P.G. Angew. Chem. Int. Ed. Engl. 1995, 34, 621; Robertson, S. A.; Ellman,J. A.; Schultz, P. G. J. Am. Chem. Soc. 1991, 113, 2722; Noren, C. J.;Anthony-Cahill, S. J.; Griffith, M. C.; Schultz, P. G. Science 1989,244, 182; Bain, J. D.; Glabe, C. G.; Dix, T. A.; Chamberlin, A. R. J.Am. Chem. Soc. 1989, 111, 8013; Bain, J. D. et al. Nature 1992, 356,537; Gallivan, J. P.; Lester, H. A.; Dougherty, D. A. Chem. Biol. 1997,4, 740; Turcatti, et al. J. Biol. Chem. 1996, 271, 19991; Nowak, M. W.et al. Science, 1995, 268, 439; Saks, M. E. et al. J. Biol. Chem. 1996,271, 23169; Hohsaka, T. et al. J. Am. Chem. Soc. 1999, 121, 34).

Transport of several analogues into the cell is indicated by a number ofliterature reports. Analogue 4 is an antagonist for methionine,inhibiting the growth of E. coli cells (Skinner, C. G.; Edelson, J.;Shive, W. J. Am. Chem. Soc. 1961, 83, 2281); 5 has been incorporatedinto proteins in E. coli cells with appropriately engineered MetRSactivity; and 8 replaces leucine in human hemoglobin expressed in E.coli (Apostol, I.; Levine, J.; Lippincott, J.; Leach, J.; Hess, E.;Glascock, C. B.; Weickert, M. J.; Blackmore, R. J. Biol. Chem. 1997,272, 28980). Although there is no similar evidence reported foranalogues 6 and 7, the fact that trifluoromethionine and ethionine areincorporated into proteins expressed in E. coli (Hendrickson, W. A.;Horton, J. R.; Lemaster, D. M. EMBO J. 1990, 9, 1665; Boles, J. O. etal. Nature Struct. Biol. 1994, 1, 283; Cowie, D. B.; Cohen, G. N.;Bolton, E. T.; de Robichon-Szulmajster, H. Biochem. Biophys. Acta 1959,34, 39; Duewel, H.; Daub, E.; Robinson, R.; Honek, J. F. Biochemistry1997, 36, 3404; Budisa, N.; Steipe, B.; Demange, P.; Eckerskorn, C.;Kellerman, J.; Huber, R. Eur. J. Biochem. 1995, 230, 788) suggests thatneither the trifluoromethyl group nor the longer side chain will inhibittransport of analogues 6 and 7 into E. coli cells.

The results of the in vitro enzyme assays shown in FIG. 5 are consistentwith the in vivo results, as the analogues that support the highestrates of PP_(i) exchange also support protein synthesis in the absenceof methionine. Although the in vitro results indicate that 5 and 11 arerecognized by MetRS, comparison of the k_(cat)/K_(m) values ofmethionine and 5 and 11 demonstrate that 5 is activated 4700-fold and 1113825-fold less efficiently than methionine; thus it is not surprisingthat neither 5 or 11 support measurable protein synthesis in the in vivoexperiments. Consideration of the in vivo and in vitro results, alongwith the reports cited earlier, suggests that transport is not limitingand that analogue incorporation is controlled by the MetRS.

Although the crystal structure of an active tryptic fragment of the E.coli MetRS (complexed with ATP) has been reported (Brunie, S.; Zelwer,C.; Risler, J. L. J. Mol. Biol. 1990, 216, 411; Mechulam, Y.; Schmitt,E.; Maveyraud, L.; Zelwer, C.; Nureki, O.; Yokoyama, S.; Konno, M.;Blanquet, S. J. Mol. Biol. 1999, 294, 1287-1297), the correspondingstructure with bound methionine is not yet available. Inferencesconcerning the mechanism of methionine (or analogue) recognition byMetRS have heretofore been made indirectly, on the basis of sequencecomparison and site-directed mutagenesis (Ghosh, G.; Pelka, H.;Schulman, L. H.; Brunie, S. Biochemistry 1991, 30, 9569; Fourmy, D.;Mechulam, Y.; Brunie, S.; Blanquet, S.; Fayat, G. FEBS Lett. 1991, 292,259; Kim, H. Y.; Ghosh, G.; Schulman, L. H.; Brunie, S.; Jakubowski, H.Proc. Natl. Acad. Sci. USA 1993, 90, 11553).

FIG. 6 compares the equipotential surfaces calculated for methionine andfor analogues 2, 3 and 5. That 2 might serve as a substrate for themethionyl-tRNA synthetase is not surprising, given the similargeometries accessible to 1 and 2, the availability of π-electrons nearthe side-chain terminus of 2, and the known translational activity ofnorleucine (9); the saturated analogue of 2. The high translationalactivity observed for 3, (i.e., near-quantitative replacement ofmethionine without loss of protein yield), was not anticipated, sincethe colinearity of side-chain carbons 4-6 imposes on 3 a geometrysubstantially different from that of methionine. However, the electrondensity associated with the triple bond of 3 is positioned similarly tothat of the thioether of the natural substrate, despite the differencesin side-chain geometry. Furthermore, given the important roles assignedto residues Phe197 and Trp305 in the E. coli methionyl-tRNA synthetase(Ghosh, G.; Pelka, H.; Schulman, L. H.; Brunie, S. Biochemistry 1991,30, 9569; Fourmy, D.; Mechulam, Y.; Brunie, S.; Blanquet, S.; Fayat, G.FEBS Lett. 1991, 292, 259; Kim, H. Y.; Ghosh, G.; Schulman, L. H.;Brunie, S.; Jakubowski, H. Proc. Natl. Acad. Sci. USA 1993, 90, 11553),alkynyl C—H/π contacts (Steiner, T.; Starikov, E. B.; Amado, A. M.;Teixeira-Dias, J. J. C. J. Chem. Soc. Perk. Trans. 2, 1995, 7, 1321) andthe polarizability of the unsaturated side chain may also playsignificant roles in recognition of 3 by the enzyme. FIG. 6 alsocompares the geometries of 1 and 5, the latter an analogue neitherrecognized efficiently by the MetRS in vitro nor translationally activein vivo. Although the geometries of 1 and 5 appear similar in therepresentation shown, the fixed planarity of the C₄-C₅ bond may precludethe side-chain conformation required for efficient recognition of 5 byMetRS. Appropriate engineering of the MetRS activities of E. coliimparts translational activity to 5.

In conclusion, a set of twelve methionine analogues was assayed fortranslational activity in Escherichia coli. Norvaline and norleucine,which are commercially available, were assayed along withhomoallylglycine (2), homopropargylglycine (3), cis-crotylglycine (4),trans-crotylglycine (5), 6,6,6-trifluoro-2-aminohexanoic acid (6) and2-aminoheptanoic acid (7) and 2-butynylglycine (11), each of which wasprepared by alkylation of diethyl acetamidomalonate with the appropriatetosylate, followed by hydrolysis. The other analogues were commerciallyavailable or prepared as described supra. The E. coli methionineauxotroph CAG18491, transformed with plasmids pREP4 and pQE15, was usedas the expression host, and translational activity was assayed bydetermination of the capacity of the analogue to support synthesis ofthe test protein dihydrofolate reductase (mDHFR) in the absence of addedmethionine.

The importance of amino acid side chain length was illustrated by thefact that neither norvaline (8) nor 7 showed translational activity, incontrast to norleucine (9), which does support protein synthesis underthe assay conditions. The internal alkene functions of 4 and 5 preventedincorporation of these analogues into test protein, and the fluorinatedanalogue 6 and 10-13 yielded no evidence of translational activity. Theterminally unsaturated compounds 2 and 3, however, proved to beexcellent methionine surrogates: ¹H NMR spectroscopy, amino acidanalysis and N-terminal sequencing indicated ca 85% substitution ofmethionine by 2, while 3 showed 90-100% replacement. Both analogues alsofunction efficiently in the initiation step of protein synthesis, asshown by their near-quantitative occupancy of the N-terminal amino acidsite in mDHFR. Enzyme kinetics assays were conducted to determine therate of activation of each of the methionine analogues by methionyl tRNAsynthetase (MetRS); results of the in vitro assays corroborate the invivo incorporation results, suggesting that success or failure ofanalogue incorporation in vivo is controlled by MetRS.

EXAMPLE II

This example demonstrates the expansion of the scope of methionineanalogues for incorporation into proteins in vivo by altering themethionyl-tRNA synthetase activity of a bacterial expression host.

The relative rates of activation of methionine and methionine analogues2-13 (FIG. 2) by MetRS were characterized in vitro by the ATP-PP_(i)exchange assay. The fully active, truncated form of MetRS was purifiedfrom overnight cultures of JM101 cells carrying the plasmid pGG3. Theenzyme was purified by size exclusion chromotography as previouslydescribed (P. Mellot, Y. Mechulam, D. LeCorre, S. Blanquet, G. Fayat, J.Mol. Biol. 1989, 208, 429-443). Activation of methionine analogues byMetRS was assayed at 25° C. via the amino acid-dependent ATP-PP_(i)exchange reaction, also as described in Example I (G. Ghosh, H. Pelka,L. H. Schulman, Biochemistry 1990, 29, 2220-2225). Assays to determineif the methionine analogues 2-13 were recognized by MetRS were conductedin solutions 75 nM in enzyme and 5 mM in the L-isomer of the analoguewith a reaction time of 20 minutes. Kinetic parameters for analogue 5were obtained with an enzyme concentration of 50 nM and analogueconcentrations of 100 μM to 10 mM. Kinetic parameters for analogue 11were determined using an enzyme concentration of 50 nM and analogueconcentrations ranging from 750 μM to 20 mM. Kinetic parameters foranalogues 4, 7, 8, and 12 were obtained with an enzyme concentration of50 or 75 nM and analogue concentrations ranging from 5 to 70 mM.Parameters for methionine were obtained by using concentrations rangingfrom 10 μM to 1 mM. K_(m) values for methionine were similar to thosepreviously reported (24±2 μM), though the measured k_(cat) was somewhatlower than the literature value (13.5 s⁻¹) (H. Y. Kim, G. Ghosh, L. H.Schulman, S. Brunie, H. Jakubowski, Proc. Natl. Acad. Sci. USA 1993, 90,11553-11557). Kinetic constants were calculated by nonlinear regressionanalysis.

FIG. 5 demonstrates that analogues 2 and 3 are activated by MetRS, asanticipated on the basis of the in vivo experiments (as described inExample I, infra; J. C. M. van Hest, D. A. Tirrell, FEBS Lett. 1998,428, 68-70), although they cause exchange of PP_(i) at ratesseveral-fold lower than methionine. Analogue 4 does not cause measurableexchange of PP_(i) by MetRS in vitro, which was expected since neither 4nor 5 were indicated to be translationally active in vivo.

Analogues 5 and 11, however, were activated by MetRS, causing slowexchange of PP_(i) under the assay conditions used herein. Table 2 showsthe k_(cat)/K_(m) values obtained for methionine, 2, 3, 5, 9, and 11.Given that k_(cat)/K_(m) for 5 is 4700-fold and that for 11 is13825-fold lower than that for methionine (as described in Example I,infra), it is not surprising that neither 5 or 11 support measurableprotein synthesis within the time frame of the in vivo experiments.

These results suggest that increasing the MetRS activity of theexpression host might allow efficient protein synthesis in culturessupplemented with 5 or 11. This strategy was not employed previously forincorporating amino acid analogues into proteins in vivo, but reports ofin vivo misacylation of tRNA substrates by overexpressed aminoacyl-tRNAsynthetase supported the viability of the approach (S. Li, N. V. Kumar,U. Varshney, U. L. RajBhandary, J. Biol. Chem. 1996, 271, 1022-1028; J.M. Sherman, M. J. Rogers, D. Soll, Nuc. Acids. Res. 1992, 20, 2847-2852;U. Varshney, U. L. RajBhandary, J. Bacteriol. 1992, 174, 7819-7826; R.Swanson, P. Hoben, M. Sumner-Smith, H. Uemura, L. Watson, D. Soll,Science 1988, 242, 1548-1551). TABLE 2 K_(CAT)/K_(M) VALUES OBTAINED FORMETHIONINE, 2, 3, 5, 9, AND 11 Analogue K_(m) (μM) k_(cat) (s⁻¹)k_(cat)/K_(m) (s⁻¹ μM⁻¹) Protein Yield, mg/L 1 24.3 ± 2   13.3 ± 0.25.47 × 10⁻¹ 35 3 2415 ± 170 2.60 ± 0.3 1.08 × 10⁻³ 35 9 4120 ± 900 2.15± 0.6 5.22 × 10⁻⁴ 20 2 4555 ± 200 1.35 ± 0.1 2.96 × 10⁻⁴ 10 5 15,675 ±250   1.82 ± 0.6 1.16 × 10⁻⁴ 0 11 38,650 ± 2000  1.51 ± 0.5 3.91 × 10⁻⁵0

Generation of Host-Vector System

A bacterial host capable of overexpressing MetRS was produced bytransforming E. coli strains B834(DE3) (Novagen, Inc., Madison, Wis.,USA), a methionine auxotroph, with repressor plasmid pREP4 andexpression plasmid pQE15-MRS (FIG. 19) (SEQ ID NO.: 1). A gene encodinga mutant MetRS was removed from plasmid pBSM547W305F (D. Fourmy, Y.Mechulam, S. Brunie, S. Blanquet, G. Fayat, FEBS Lett. 1991, 292,259-263) by treatment with restriction enzymes Sac I and Kpn I. The SacI/Kpn I fragment (2450 bp) was ligated into the cloning vectorpUC19-Nhelink, which was constructed to permit the cohesive ends of themutant MetRS gene to be changed to Nhe I. The MetRS gene with Nhe Icohesive ends was then ligated into the unique Nhe I site of the plasmidpQE15 (Qiagen, Inc., Santa Clarita, Calif., USA) to yield plasmidpQE15-W305F (FIG. 20) (SEQ ID NO.: 2). Transformation of pQE15-W305F(SEQ ID NO.: 2) into a recA positive cell strain resulted in geneticrecombination of the mutant MetRS gene with the chromosomal copy of thewild-type MetRS gene, yielding plasmid pQE15-MRS (SEQ ID NO.: 1).

Expression plasmid pQE15-MRS (SEQ ID NO.: 1) and repressor plasmid pREP4were transformed into the expression host B834(DE3) to yieldB834(DE3)/pQE15-MRS/pREP4. Plasmid DNA from allB834(DE3)/pQE15-MRS/pREP4 cultures used for protein expressionexperiments was sequenced to confirm that it encoded wild-type MetRS.The expression plasmid pQE15-MRS (SEQ ID NO.: 1) encodes MetRS undercontrol of the E. coli promoter metG p1 (Genbank accession numberX55791) (F. Dardel, M. Panvert, G. Fayat, Mol. Gen. Genet. 1990, 223,121-133) as well as the target protein murine dihydrofolate reductase(mDHFR) under control of a bacteriophage T5 promoter. The expressionplasmid also encodes an N-terminal hexahistidine sequence for mDHFRwhich permits purification of the target protein by immobilized metalchelate affinity chromatography (The Qiagen Expressionist, 1992, p. 45).Furthermore, mDHFR contains 8 methionine residues which can be replacedby methionine analogues. A control bacterial host, which produces onlymDHFR and normal cellular levels of MetRS, was prepared by transformingB834(DE3) with pREP4 and pQE15.

Similarly, a bacterial host capable of overexpressing MetRS was producedby transforming E. coli strains CAG18491 (Novagen, Inc., Madison, Wis.,USA), a methionine auxotroph, with repressor plasmid pREP4 andexpression plasmid pQE15-MRS, as described for the B834(DE3) strain. Acontrol bacterial host, which produces only mDHFR and normal cellularlevels of MetRS, was prepared by transforming CAG18491 with pREP4 andpQE15.

Methionine analogues 2-13 were tested for translational activity in bothbacterial hosts. Methionine analogues were synthesized via alkylation ofdiethylacetamidomalonate, as previously described (As described inExample I, infra; J. C. M. van Hest, D. A. Tirrell, FEBS Lett. 1998,428, 68-70). Cultures of B834(DE3)/pQE15-MRS/pREP4 orB834(DE3)/pQE15/pREP4 or CAG18491/pQE15-MRS/pREP4 orCAG18491/pQE15/pREP4 in M9AA media were grown to an optical density of0.90, and the cells were sedimented by centrifugation. The M9AA mediumwas prepared by supplementing sterile M9 medium with 60 mg/ml of each ofthe amino acids, 1 mM MgSO₄, 0.2 wt % glucose, 1 mg/ml thiaminechloride, and 1 mg/ml calcium chloride. The antibiotics ampicillin andkanamycin were added at concentrations of 200 mg/l and 35 mg/l,respectively.

Cells were washed three times with M9 salts and resuspended to anoptical density of 0.90 in M9 test media containing 19 amino acidsplus 1) neither methionine nor analogue (negative control); 2)methionine (60 mg/liter, positive control); or 3) an analogue ofinterest (60 mg/liter). To test the effect of increasing the level ofsupplementation of the analogues, a set of experiments was alsoconducted in which the medium was supplemented with 500 mg/liter ofmethionine or the amino acid analogue. Expression of mDHFR was inducedby addition of 0.4 mM isopropyl β-D-thiogalactopyranoside (IPTG), andprotein synthesis was monitored after 4.5 hours. Expression of mDHFR wasmonitored by SDS-polyacrylamide gel electrophoresis (SDS-PAGE);accumulation of target protein was taken as evidence for translationalactivity of the methionine analogue.

For cultures supplemented with amino acids at 60 mg/liter, the targetprotein was not observed in the negative control culture ofB834(DE3)/pQE15/pREP4, CAG18491/pQE15/pREP4 or in cultures supplementedwith Ccg (4), 6,6,6-trifluoro-2-aminohexanoic acid (6), 2-aminoheptanoicacid (7), norvaline (8) o-allylserine (10), allylgylcine (12) orpropargylglycine (13). In contrast, mDHFR was detected in both bacterialhost cultures supplemented with methionine (1), Hag (2), Hpg (3), andnorleucine (9), as indicated by the appearance of a protein band at theposition expected for mDHFR in SDS-PAGE.

For the negative control cultures and for cultures supplemented withTcg, however, the behavior of the bacterial hosts differed, as shown inFIG. 7. mDHFR was not detected in the B834(DE3)/pQE15/pREP4 culturesupplemented with Tcg, while strong induction of mDHFR was observed forB834(DE3)/pQE15-MRS/pREP4 under the same conditions. Even theunsupplemented control culture of B834(DE3)/pQE15-MRS/pREP4 showsevidence of mDHFR synthesis, suggesting that introduction of pQE15-MRS(SEQ ID NO.: 1) does indeed increase the rate of activation ofmethionine in the modified host.

B834(DE3)/pQE15-MRS/pREP4 cells, which overexpress MetRS, havesufficient MetRS activity to synthesize measurable levels of proteinfrom the very low intracellular levels of methionine in the negativecontrol culture. Interestingly, aminoacyl-tRNA synthetase overexpressionis induced by amino acid starvation in some gram-positive bacteria,presumably to permit continued protein synthesis (D. Luo, J. Leautey, M.Grunberg-Manago, H. Putzer, J. Bacteriol. 1997, 179, 2472-2478).B834(DE3)/pQE15/pREP4 cultures, which lack the increased MetRS activity,do not show background expression of protein in negative controlcultures.

Similar results were observed for the CAG18491/pQE15/pREP4 andCAG18491/pQE15-MRS/pREP4 cultures supplemented with 11 (FIG. 8). WhilemDHFR was not detected in the CAG18491/pQE15/pREP4 cultures supplementedwith 11, strong induction of mDHFR was observed forCAG18491/pQE15-MRS/pREP4 under the same conditions. The unsupplementedcontrol culture of CAG18491/pQE15-MRS/pREP4 showed little evidence ofmDHFR synthesis, which may be due to lower levels of MetRS activity inthese cell strains versus that in the B 834(DE3)/pQE15-MRS/pREP4.

For cultures supplemented with amino acids at 500 mg/liter, however, thetarget protein mDHFR could be observed for certain amino acid analoguesonly in cultures of cellular hosts containing the MetRS (FIG. 9). FIG. 9demonstrates that the modified bacterial hosts CAG18491/pQE15-MRS/pREP4are able to produce the target protein in cultures supplemented with 500mg/liter of 4, 7, 8, and 12. Quantitative characterization of thekinetic parameters of these analogues demonstrates that although theanalogues do not support measurable levels of PP_(i) exchange after 20minutes, they are activated by the MetRS in vitro (FIG. 10). Due to thevery slow rate of activation supported by these analogues, increasingthe concentration of the analogues in the medium must be combined withintroduction of pQE15-MRS (SEQ ID NO.: 1) into the bacterial host inorder to raise the rate of activation of these analogues sufficiently topermit protein biosynthesis.

To confirm the supposition that the MetRS activity of a cellular host isincreased by the introduction of pQE15-MRS, direct measurement of theMetRS activities of whole cell lysates was conducted.B834(DE3)/pQE15-MRS/pREP4 exhibits a V_(max) for methionine activationapproximately 30-fold higher than that observed for the control hostB834(DE3)/pQE15/pREP4 (FIG. 11). Similarly, CAG18491/pQE15-MRS/pREP4exhibits a V_(max) for methionine activation approximately 50-foldhigher than that observed for the control host CAG18491/pQE15/pREP4.ATP-PP_(i) exchange assays were conducted using the methods as describedsupra. A 50-μl aliquot of whole cell lysate with a normalized OD₆₀₀ of20 was prepared by one freeze-thaw cycle and added to the assay mixtureto yield a final volume of 150 μl. A saturating concentration ofmethionine (750 μM) was used to determine the maximum exchange velocityfor each cell lysate.

These results show clearly that increasing the MetRS activity of thehost is necessary and sufficient to observe translational activity of 4,5, 7, 8, 11, and 12 under convenient conditions in vivo. Protein yields(mDHFR-Tcg) of approximately 8.5 mg/liter were observed forB834(DE3)/pQE15-MRS/pREP4 cultures supplemented with Tcg, compared withyields of approximately 35 mg/liter for both B834(DE3)/pQE15-MRS/pREP4and B834(DE3)/pQE15/pREP4 cultures supplemented with methionine. Aminoacid analysis of protein containing Tcg shows a decrease in methioninecontent to 0.3 mol % from the expected value of 3.8 mol %. It was notpossible to detect Tcg directly by amino acid analysis, owing toinstability of the analogue under the analysis conditions. If depletionof methionine is assumed to result from replacement by Tcg, the observedanalysis corresponds to an overall extent of incorporation of theanalogue of 91±2%. Amino acid analysis of mDHFR containing the otheranalogues (4, 7, 8, 11, and 12), showed 92-98% replacement ofmethionine.

A direct assessment of the extent of incorporation of Tcg into mDHFR wasprovided by NMR spectroscopy. Proton NMR spectra were recorded using aVarian Inova NMR spectrometer with proton acquisition at 599.69 MHz.Spectra were recorded at 25° C. overnight. A simple presaturation pulsewas used for water suppression. Comparisons of the 600 MHz proton NMRspectra (FIG. 12) of mDHFR, Tcg, and mDHFR-Tcg indicate the appearance,in the mDHFR-Tcg spectrum (FIG. 12 c), of the Tcg vinylene protons atδ=5.35 (δ-CH) and δ=5.60-5.70 (γ-CH). The resonances at δ=5.35 andδ=5.70 occur at the same chemical shift values as in free Tcg and areclearly due to incorporation of Tcg into mDHFR. That the resonance atδ=5.60 arises from the γ-CH vinylene proton of Tcg is suggested by thefact that the integrated intensity of the resonance at δ5.35 equals thesum of the integrations of the resonances at δ=5.60 and δ=5.70. Thisassignment is confirmed by ID TOCSY (Total Correlation Spectroscopy)experiments which indicate that the protons at both δ=5.60 and δ=5.70are members of the same spin system (and therefore the same amino acid)as those at δ=5.35. More importantly, the 1D TOCSY experiments also showthat the protons at δ=5.35 (and therefore those at δ=5.60 and δ=5.70)are associated with the spin system of the entire Tcg side chain (1DTOCSY spectra were recorded on a Varian Inova NMR spectrometer withproton acquisition at 599.69 MHz).

A 1D TOCSY pulse sequence (D. Uhrin, P. N. Barlow, J. Magn. Reson. 1997,126, 248-255) with selective irradiation of the resonance at δ=5.35 (E.Kupce, J. Boyd, I. D. Campbell, J. Magn. Reson. Ser. B. 1995, 106,300-303) was used to identify which protons belonged to the spin systemof the δ=5.35 resonance. The selectivity of the pulse is demonstrated ina separate, simple 1D experiment in which the selective pulse wasapplied alone; no other resonances were observed in the spectrum underthese conditions. Observation after a mixing time of 60 ms, however,showed the protons at δ=5.60 and δ=5.70, indicating that, those protonsare members of the same spin system (and therefore the same amino acidresidue) as those corresponding to the resonance at δ=5.35. The α-carbonand side chain β- and ε-carbon protons were also observed at chemicalshift values characteristic of the free amino acid (ε4.3 (α-CH), 2.5(β-CH2), and 1.6 (δ-CH3)). Integration of the spectrum suggests that 5of the 8 methionine positions (occupied by Tcg) are represented by theresonance at δ=5.60; these protons must reside in amagnetically-distinct environment from the protons at δ=5.70. Theseresults unequivocally demonstrate the translational activity of Tcg inthe host strain outfitted with elevated MetRS activity. Integration ofthe NMR spectrum indicates 90±6% replacement of methionine.

Retention of the N-terminal (initiator) methionine in mDHFR was expectedon the basis of the identity of the penultimate amino acid (P. H. Hirel,J. M. Schmitter, P. Dessen, G. Fayat, S. Blanquet, Proc. Natl. Acad.Sci. USA 1989, 86, 8247-8251), so N-terminal sequencing provided a thirdmeans of assessing the extent of replacement of methionine by analogues4, 5, 7, 8, 11, and 12. Because the analogues were not degraded underthe analysis conditions, they could be detected directly. Comparison ofchromatograms of the N-terminal residues of mDHFR and mDHFR-Tcg (FIG.13) demonstrate that the methionine that normally occupies the initiatorposition of mDHFR (FIG. 13 a) was nearly completely replaced with Tcg(FIG. 13 b) in mDHFR-Tcg (FIG. 13 c). The signal corresponding tomethionine eluted at 13.8 min while that corresponding to Tcg eluted at16.0 min. The large peaks (pptu) which elute at approximately 15.4 mincorrespond to piperidylphenylthiourea, a product of the analysisresulting from the buffer, and the small peak (diet) at 19.4 mincorresponds to diethylphthalate, an internal standard. These resultsclearly indicate the incorporation of Tcg at the initiator site ofmDHFR-Tcg and corroborate the NMR results. Integration of the peak areascorresponding to Tcg and to methionine indicates 96±2% incorporation ofthe analogue at the initiator position. Similar analysis of mDHFR-2bg(2-butynylglycine) by N-terminal sequencing indicates 98±2% replacementof methionine (FIG. 14). Analysis of mDHFR containing analogues 8 and 12shows replacement of methionine by these analogues at levels of 85-90%.

The incorporation of 4, 5, 7, 8, 11, and 12 into proteins in vivoconstitutes the first example of broadening the amino acid substraterange of the E. coli translational apparatus via overproduction of MetRSin a bacterial host. The utilization of 4, 5, 7, 8, 11, and 12 in allstages of protein synthesis (including initiation) indicates theappropriateness of targeting the aminoacyl-tRNA synthetase in studiesaimed at in vivo incorporation of amino acid analogues into proteins.Transport into the cell, recognition by methionyl-tRNA formylase, andrecognition by the elongation factors and the ribosome are less likelyto be limiting factors.

These results indicate that this simple strategy of overexpression ofaminoacyl-tRNA synthetase may be used to modify proteins byincorporation of amino acid analogues that are poor substrates foraminoacyl-tRNA synthetase and that would be essentially inactive inconventional expression hosts.

Overexpression of mutant forms of the aminoacyl-tRNA synthetase preparedvia site-directed mutagenesis or directed evolution (F. H. Arnold, J. C.Moore, Adv. Biochem. Eng. Biotech. 1997, 58, 1-14; F. H. Arnold, Chem.Eng. Sci. 1996, 51, 5091-5102) should provide additional strategies forincorporating amino acid analogues into proteins in vivo.

The results reported here also suggest new opportunities formacromolecular synthesis via protein engineering. The versatilechemistry of unsaturated functional groups (B. M. Trost, I. Fleming,Comprehensive Organic Synthesis, Pergamom Press, Oxford, 1991) can beused to control protein structure and function through chemicalderivatization, an especially intriguing possibility in this case giventhe important role of methionine in protein-protein recognitionprocesses. For example, ruthenium-catalyzed olefin metathesis (D. M.Lynn, B. Mohr, R. H. Grubbs, J. Am. Chem. Soc. 1998, 120, 1027-1028; E.L. Dias, T. N. SonBinh, R. H. Grubbs, J. Am. Chem. Soc. 1997, 119,3887-3897) of homoallylglycine (T. D. Clark, M. R. Ghadiri, J. Am. Chem.Soc. 1995, 117, 12364-12365) and o-allylserine (H. E. Blackwell, R. H.Grubbs, Angew. Chem. 1998, 110, 3469-3472; Angew. Chem. Int. Ed. 1998,37, 3281-3284) side chains has been used to produce covalently-modifiedpeptide structures of various kinds. The incorporation of Tcg may besingularly useful in this regard as the internal olefin is active inaqueous-phase ring closing metathesis reactions, whereasterminally-unsaturated groups (such as those previously used to replacemethionine in vivo) are not (T. A. Kirkland, D. M. Lynn, R. H. Grubbs,J. Org. Chem. 1998, 63, 9904-9909).

EXAMPLE III

This example demonstrates that activation of methionine analogues invitro correlates well with the ability of these analogues to supportprotein synthesis in vivo, substantiating the critical role ofaminoacyl-tRNA synthetase in controlling the incorporation of amino acidanalogues into proteins.

Reagents

Each of the analogues 2-7 and 11 (FIG. 2) was prepared by alkylation ofdiethyl acetamidomalonate with the appropriate tosylate followed bydecarboxylation and deprotection of the amine function (as described inExample I). Methionine and analogues 8, 9, 12 and 13 were obtained fromSigma (St. Louis, Mo.). Radiolabeled sodium pyrophosphate was purchasedfrom NEN Life Science Products, Inc., andisopropyl-β-D-thiogalactopyranoside was obtained from Calbiochem. TheRGS-His antibody and anti-mouse IgG horseradish peroxidase conjugateused for Western blotting procedures were obtained from Qiagen andAmersham Life Sciences, respectively. All other reagents used duringprotein biosynthesis and purification and for activation assays werecommercially available from Sigma, Aldrich, and Qiagen, and were used asreceived.

In Vitro Activation Assays

The fully active, truncated form of MetRS was purified from overnightcultures of E. coli JM101 cells carrying the plasmid pGG3 (Kim, H. Y.;Ghosh, G.; Schulman, L. H.; Brunie, S.; Jakubowski, H. Proc. Natl. Acad.Sci. USA 1993, 90, 11553-11557), by using size exclusion methodspreviously reported (Mellot, P.; Mechulam, Y.; LeCorre, D.; Blanquet,S.; Fayat, G. J. Mol. Biol. 1989, 208, 429-443). Purified enzymesolutions (in 10 mM phosphate, pH 6.7, 10 mM (β-mercaptoethanol) wereconcentrated to at least 3 μM prior to their storage in 40% glycerol at−20° C. Concentrations of enzyme stocks were determined by the Bradfordmethod, using samples of MetRS quantified by amino acid analysis asstandards.

Activation of methionine analogues by MetRS was assayed via the aminoacid-dependent ATP-PP_(i) exchange reaction at room temperature, also aspreviously described (Mellot, P.; Mechulam, Y.; LeCorre, D.; Blanquet,S.; Fayat, G. J. Mol. Biol. 1989, 208, 429-443; Ghosh, G.; Pelka, H.;Schulman, L. D. Biochemistry 1990, 29, 2220-2225). The assay, whichmeasures the ³²P-radiolabeled ATP formed by the enzyme-catalyzedexchange of ³²P-pyrophosphate (PP_(i)) into ATP, was conducted in 150 μlof reaction buffer (pH 7.6, 20 mM imidazole, 0.1 mM EDTA, 10 mMβ-mercaptoethanol, 7 mM MgCl₂, 2 mM ATP, 0.1 mg/ml BSA, and 2 mM PP_(i)(in the form of sodium pyrophosphate with a specific activity ofapproximately 0.5 TBq/mole)).

Kinetic parameters for methionine analogues 2, 3, 5, 9 and 11 wereobtained with an enzyme concentration of 75 nM and analogueconcentrations of 100 μM to 20 mM. Parameters for methionine wereobtained by using methionine concentrations ranging from 10 μM to 1 Mm.Aliquots (20 μl) were removed from the reaction mixture at various timepoints and quenched in 0.5 ml of a solution comprising 200 mM PP_(i), 7%w/v HClO₄, and 3% w/v activated charcoal. The charcoal was rinsed twice,with 0.5 mL of a 10 mM PP_(i), 0.5% HClO₄ solution and then resuspendedin 0.5 mL of this solution and counted via liquid scintillation methods.Kinetic constants were calculated by a nonlinear regression fit of thedata to the Michaelis-Menten model.

In Vivo Incorporation of Amino Acid Analogues

Buffers and media were prepared according to standard protocols(Ausubel, F. M.; Brent, R.; Kingston, R. E.; Moore, D. D.; Seidman, J.G.; Smith, J. A.; Struhl, K., Eds. Current Protocols in MolecularBiology; John Wiley and Sons: New York, 1998). The E. coli methionineauxotroph CAG18491 (λ⁻, rph-1, metE3079.:Tn10) was transformed withplasmids pQE15 and pREP4 (Qiagen), to obtain the expression hostCAG18491/pQE15/pREP4. The auxotroph was transformed with the plasmidspQE15-MRS (SEQ ID NO.: 1) and pREP4 to obtain the modified bacterialexpression host CAG18491/pQE15-MRS/pREP4. Both bacterial expressionhosts produce the target protein mDHFR under control of a bacteriophageT5 promoter; the modified host also expresses extra copies of the MetRSgene under control of the constitutive metG p1 promoter (Dardel, F.;Panvert, M.; Fayat, G. Mol. Gen. Genet. 1990, 223, 121.133).

Protein Expression (1 liter Scale).

Similar procedures were used for preparation and isolation of mDHFR frommedia supplemented with the L-isomers of 1, 2, 3, or 9. M9AA medium (100mL) supplemented with 1 mM MgSO₄ 0.2 wt % glucose, 1 mg/L thiaminechloride and the antibiotics ampicillin (200 mg/L) and kanamycin (35mg/L) was inoculated with the appropriate E. coli strain(CAG18491/pQE15/pREP4 or CAG18491/pQE15-MRS/pREP4) and grown overnightat 37° C. This culture was used to inoculate 900 mL M9AA mediumsupplemented as described. The cells were grown to an optical density at600 nm (OD₆₀₀) of approximately 0.9 and a medium shift was performed.The cells were sedimented for 10 min at 3030 g at 4° C., the supernatantwas removed, and the cell pellet was washed twice with 600 mL of M9medium. Cells were resuspended in 1000 mL of the M9AA medium describedabove, without methionine, and supplemented with 20 mg/L of the L-isomerof either 1, 2, 3, or 9. Protein synthesis was induced by addition ofisopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of0.4 mM. Samples (1 mL) were collected after 4.5 hours, the OD₆₀₀measured, and cells resuspended with distilled water to yield anormalized OD₆₀₀ of 20. Protein expression was monitored by SDSpolyacrylamide gel electrophoresis (12% acrylamide running gel);accumulation of mDHFR could be observed at an apparent molar mass ofapproximately 28 kDa after Coomassie staining.

Protein Purification.

Approximately 4.5 h after induction, cells were sedimented (9,800 g, 10min, 4° C.) and the supernatant was removed. The pellet was placed inthe freezer overnight. The cells were thawed for 30 min at 37° C., 30 mLof buffer (6 M guanidine-HCl, 0.1 M NaH₂PO₄, 0.01 M Tris, pH 8) wasadded and the mixture was shaken at room temperature for 1 h. The celldebris was sedimented (15,300 g, 20 min, 4° C.) and the supernatant wassubjected to immobilized metal affinity chromatography (Ni-NTA resin)according to the procedure described by Qiagen (The QiagenExpressionist; Qiagen; Valencia, Calif., 2000). The supernatant wasloaded on 10 mL of resin which was then washed with 50 mL of guanidinebuffer followed by 25 mL of urea buffer (8 M urea, 0.1 M NaH₂PO₄ and0.01 M Tris, pH 8). Similar urea buffers were used for three successive25 mL washes at pH values of 6.3, 5.9 and 4.5, respectively. Targetprotein was obtained in washes at pH 5.9 and 4.5. These washes werecombined and dialyzed (Spectra/Por membrane 1, MWCO=6-8 kDa) bybatchwise dialysis against doubly distilled water for 4 days with atleast 12 total changes of water. The dialysate was lyophilized to apurified powder of mDHFR. Experiments in M9AA medium affordapproximately 30 mg of mDHFR for each of the bacterial expression hosts,while a control experiment in 2×YT medium afforded approximately 60 mgof mDHFR. Protein yields are reported as mg protein obtained per literof bacterial culture; approximately 5-6 g of wet cells are obtained perliter of culture regardless of the identity of the analogue used tosupplement the medium.

Protein Expression (5 mL Scale).

M9AA medium (50 mL) supplemented with 1 mM MgSO₄, 0.2 wt % glucose, 1mg/L thiamine chloride and the antibiotics ampicillin (200 mg/L) andkanamycin (35 mg/L) was inoculated with 5 mL of an overnight culture ofthe appropriate bacterial expression host. When the turbidity of theculture reached an OD₆₀₀ of 0.8, a medium shift was performed. The cellswere sedimented for 10 min at 3030 g at 4° C., the supernatant wasremoved, and the cell pellet was washed twice with 25 mL of M9 medium.Cells were resuspended in 50 mL of the M9AA medium described above,without methionine. Test tubes containing 5 mL aliquots of the resultingculture were prepared, and were supplemented with 10 μL of 10 mg/mLL-methionine (1) (positive control), L-homoallylglycine (2),L-homopropargylglycine (3), or L-norleucine (9), respectively. A culturelacking methionine (or any analogue) served as the negative control.Protein expression was induced by addition of IPTG to a finalconcentration of 0.4 mM. After 4 h, the OD₆₀₀ was measured, and thesamples were sedimented. After the supernatant was decanted, the cellpellets were resuspended in distilled water to yield a normalized OD of20.

Protein expression was monitored by SDS polyacrylamide gelelectrophoresis (12% acrylamide running gel), followed by Westernblotting. After transfer to a nitrocellulose membrane, Western blotswere developed by treatment with a primary RGS-His antibody, followed bytreatment with a secondary anti-mouse IgG conjugated to horseradishperoxidase to provide detection by chemiluminescence. Films were checkedto ensure that band intensity was not saturated. Levels of proteinsynthesis were estimated by the intensity of the band on the gel, asdetermined using a Pharmacia Ultrascan XL laser densitometer andanalysis by Pharmacia GelScan XL software. The accumulation of targetprotein is taken as evidence for incorporation of the amino acidanalogue, as 2, 3, 5, 9 and 11 have been shown to replace methionine,even in modified bacterial hosts, at levels of 92-98% (van Hest, J. C.M.; Tirrell, D. A. FEBS Lett. 1998, 428, 68-70; as described in ExampleI, infra; Budisa, N.; Steipe, B.; Demange, P.; Eckerskorn, C.;Kellermann, J.; Huber, R. Eur. J. Biochem. 1995, 230, 788-796).

Results and Discussion

Studies with methionine analogues 2-13 (as described in Example I,infra), demonstrated that 2 and 3 can be incorporated into proteins withextents of substitution up to 98%. The incorporation of 9 had beenpreviously reported (Budisa, N.; Steipe, B.; Demange, P.; Eckerskorn,C.; Kellermann, J.; Huber, R. Eur. J. Biochem. 1995, 230, 788-796). Incontrast, 4-8 and 10-13 do not support protein synthesis in the absenceof methionine in a conventional bacterial expression host; investigationof the activation of the analogues by methionyl-tRNA synthetase (MetRS)indicated that 4-8 and 10-13 are not efficiently activated by theenzyme.

Overproduction of MetRS in the bacterial host, however, permitsincorporation of 4, 5, 7, 8, 11 and 12, which show very slow exchange ofPP_(i) in in vitro activation assays (Example II, infra). These resultsindicate that the aminoacyl-tRNA synthetase are appropriate targets forstudies aimed at the incorporation of amino acid analogues into proteinsin vivo. The results also suggest that neither transport into the cellnor recognition by the elongation factors or the ribosome limits theincorporation of these amino acid analogues into proteins in vivo.

In this example, the in vitro activation of 2-13 by MetRS wascharacterized in order to determine the roles of the synthetase incontrolling analogue incorporation and protein yield in mediasupplemented with amino acid analogues. Furthermore, the analogues 2 and3, which replace methionine in vivo, may be useful for chemicalmodification of proteins by olefin metathesis (Clark, T. D.; Kobayashi,K.; Ghadiri, M. R. Chem. Eur. J. 1999, 5, 782-792; Blackwell, H. E.;Grubbs, R. H. Angew. Chem. Int. Ed. Engl. 1998, 37, 3281-3284),palladium-catalyzed coupling (Amatore, C.; Jutand, A. J. Organomet.Chem. 1999, 576, 255-277; Tsuji, J. Palladium Reagents and Catalysts:Innovations in Organic Synthesis; John Wiley and Sons: New York, 1995;Schoenberg, A.; Heck, R. F. J. Org. Chem. 1974, 39, 3327-3331), andother chemistries (Trost, B. M.; Fleming, I., Eds. Comprehensive OrganicSynthesis; Pergamon Press: Oxford, 1991).

The attachment of an amino acid to its cognate tRNA proceeds in twosteps (FIG. 18). Activation, the first step, involves theenzyme-catalyzed formation of an aminoacyl adenylate (designated aa˜AMPin FIG. 18) and can be examined by monitoring the rate of exchange ofradiolabeled pyrophosphate (³²P-PP_(i)) into ATP (Fersht, A. Structureand Mechanism in Protein Science; W.H. Freeman and Company: New York,1999). Aminoacylation, the second step, can be evaluated by monitoringthe amount of radiolabeled amino acid attached to tRNA in the presenceof the enzyme. Because initial recognition of an amino acid by itsaminoacyl-tRNA synthetase is perhaps the most critical step in theincorporation of amino acid analogues into proteins in vivo. Thus, thefocus has been on the in vitro activation of methionine analogues byMetRS was evaluated and the results compared to those obtained instudies of in vivo incorporation.

The rates of activation of 2-13 by MetRS were determined by theATP-PP_(i) exchange assay, and were found to correlate well with theresults of the in vivo studies; analogues 2-5, 7-9, 11 and 12 (thosewhich had been shown to support protein synthesis) exhibited measurableexchange of PP_(i) (Examples I and II, infra). The kinetic parametersk_(cat) and K_(m) were determined for each of these analogues; theresults for analogues 2, 3, 5, 9 and 11 are summarized in FIG. 15. Themeasured K_(m) for methionine matched previously reported values (Kim,H. Y.; Ghosh, G.; Schulman, L. H.; Brunie, S.; Jakubowski, H. Proc.Natl. Acad. Sci. USA 1993, 90, 11553-11557). The value determined fork_(cat) was slightly lower than the literature value. Comparison of thek_(cat)/K_(m) values for each of the analogues with that for methionineshowed that these analogues were 500-fold to 13825-fold poorersubstrates for MetRS than methionine.

FIG. 15 also demonstrates that methionine analogues that are activatedup to 2000-fold more slowly by MetRS than methionine can support proteinsynthesis in a conventional bacterial host in the absence of methionine.(Poorer substrates, such as 5, require modification of the MetRSactivity of the bacterial host in order to support protein synthesis(Example II, infra). These results are comparable to those reportedpreviously for the activation and in vivo incorporation of phenylalanineanalogues (Gabius, H. J.; von der Haar, F.; Cramer, F. Biochemistry1983, 22, 2331-2339; Kothakota, S.; Mason, T. L.; Tirrell, D. A.;Fournier, M. J. J. Am. Chem. Soc. 1995, 117, 536-537; Ibba, M.; Kast,P.; Hennecke, H. Biochemistry 1994, 33, 7107-7112). Comparisons forother amino acids have been limited by a lack of in vitro activationdata. The data suggested that amino acid analogues can support proteinsynthesis in vivo even with surprisingly inefficient activation of theamino acid by its aminoacyl-tRNA synthetase. Activation of methionineanalogues by MetRS governs their ability to support protein synthesis invivo.

Based on these results, it seemed likely that the kinetics of analogueactivation would limit the rate and yield of protein synthesis inbacterial cultures supplemented with methionine analogues that are poorsubstrates for MetRS. This correlation was investigated by comparing thekinetic constants for analogue activation by MetRS with the yield of thetarget protein murine dihydrofolate reductase (mDHFR) obtained from1-liter cultures of the bacterial host CAG18491/pQE15/pREP4.

The CAG18491/pQE15/pREP4 bacterial host was produced by transforming theE. coli methionine auxotroph CAG18491 with the expression plasmid pQE15and the repressor plasmid pREP4. The expression plasmid pQE15 encodesmDHFR under control of a bacteriophage T5 promoter and an N-terminalhexahistidine sequence that permits purification of the target proteinby immobilized metal chelate affinity chromatography (The QiagenExpressionist; Qiagen; Valencia, Calif., 2000).

The kinetic constants for analogue activation and the correspondingprotein yields are listed in FIG. 15 and shown in FIG. 16. Analogueswith the highest k_(cat)/K_(m) values also support the highest levels ofprotein synthesis; the protein yields scale remarkably well withk_(cat)/K_(m), at least for the poorer substrates. Analogue 3 supportedprotein synthesis with yields equivalent to those obtained withmethionine, despite the fact that 3 is a 500-fold poorer substrate forMetRS than methionine.

Bacterial cultures supplemented with 9 (1050-fold lower k_(cat)/K_(m))produce 57% as much mDHFR as cultures supplemented with methionine, andcultures supplemented with 2 (1850-fold lower k_(cat)/K_(m)) produce 28%of the control yield of protein. Bacterial cultures supplemented with 5(4700-fold lower k_(cat)/K_(m)) and 11 (13825-fold lower k_(cat)/K_(m))did not support measurable levels of protein synthesis in thisexpression host; however, bacterial hosts exhibiting approximately30-fold higher MetRS activity produce 23% as much mDHFR in culturessupplemented with 5 as cultures supplemented with methionine (ExampleII, infra).

These results demonstrate that the rate of methionine analogueactivation in vitro does indeed correlate with protein yield in vivo.The results suggest that the kinetics of activation can play a criticalrole in controlling the rate of protein synthesis in methionine-depletedcultures supplemented with analogues that are poor substrates for MetRS.

Protein yields obtained from bacterial cultures supplemented withmethionine analogues might be improved by increasing the MetRS activityof the bacterial host. To test this, the yields of protein prepared werecompared in the conventional bacterial expression host,CAG18491/pQE15/pREP4, to those obtained from a modified host,CAG18491/pQE15-MRS/pREP4.

The modified CAG18491/pQE15-MRS/pREP4 host was prepared-by transformingE. coli strain CAG18491 with the expression plasmid pQE15-MRS (SEQ IDNO.: 1) (Example II, infra) and the repressor plasmid pREP4. Theexpression plasmid pQE15-MRS (SEQ ID NO.: 1) encodes MetRS under controlof the E. coli promoter metG p1 (Genbank accession number X55791)(Dardel, F.; Panvert, M.; Fayat, G. Mol. Gen. Genet. 1990, 223, 121-133)as well as the target protein mDHFR. The MetRS activity of the bacterialhosts was determined as previously described (Example II, infra), withthe modified host exhibiting 50-fold higher MetRS activity than theconventional strain.

Protein synthesis was monitored for 5-ml cultures of these hostssupplemented with methionine or analogues 2, 3, or 9. Western blotanalyses of protein synthesis are shown in FIG. 17. Although very lowlevels of protein synthesis were observed for negative control culturesof CAG18491/pQE15-MRS/pREP4, amino acid analyses, N-terminal sequencing,and NMR analyses of proteins produced in cultures of the modified hostsupplemented with 5 and 11 (the poorest of the substrates) still showed90-96% replacement of methionine by 5 and 11 (Example II, infra). Thus,the level of protein synthesis shown in FIG. 17 resulted from theincorporation of the analogue and was not due to incorporation ofresidual methionine.

For cultures supplemented with methionine or 3, the modified host,CAG18491/pQE15-MRS/pREP4, does not exhibit higher levels of proteinsynthesis than the conventional host CAG18491/pQE15/pREP4. Analysis bylaser densitometry confirmed these results, and revealed approximatelyequal accumulation of target protein for both strains; identical resultshave been obtained for large-scale expressions and purification ofmDHFR. Activation of the analogue by MetRS does not appear to limitprotein synthesis in cultures supplemented with 3. For culturessupplemented with 2 or 9, however, the modified bacterial host exhibitssignificantly increased levels of protein synthesis in comparison withthe conventional host. Laser densitometry analysis indicates that thelevel of protein synthesis in the modified host is increasedapproximately 1.5-fold over that in the conventional host for culturessupplemented with 2, and approximately 1.4-fold for culturessupplemented with 9. Activation of these analogues by MetRS appears tolimit protein synthesis in the conventional host, such that increasingthe MetRS activity of the host is sufficient to restore high levels ofprotein synthesis. Preliminary results indicated that the yield of mDHFRobtained from large-scale cultures of CAG18491/pQE15-MRS/pREP4supplemented with 2 or 9 are increased to approximately 35 mg/L (from 10mg/L obtained from cultures of CAG18491/pQE15/pREP4 (FIG. 15)).

The results indicate that overexpression of MetRS can improve proteinyields for cultures supplemented with methionine analogues that are poorsubstrates for MetRS, and provide an attractive general method forefficient production of chemically novel protein materials in vivo.

Quantitative assessment of the kinetics of activation by MetRS haveindicated that even very poor substrates for the synthetase can beutilized by the protein synthesis machinery of a bacterial expressionhost. The correlation, shown herein, between the in vitro and in vivoresults indicates the important role of the aminoacyl-tRNA synthetaseand suggests that site-directed mutagenesis and/or directed evolution ofthis class of enzymes may be used to increase further the number ofamino acid analogues that can be incorporated into proteins in vivo.

These results also indicate that the kinetics of activation ofmethionine analogues by MetRS in vitro correlate with the level ofprotein synthesis supported by the analogues in vivo. The activity ofthe MetRS in the bacterial host can be manipulated, by overexpression ofthe MetRS, to improve the yields of proteins containing methionineanalogues that are poor substrates for the MetRS. Overexpression ofaminoacyl-tRNA synthetase used to improve yields of proteins containingother amino acid analogues, as well as proteins rich in particularnatural amino acids. Manipulation of the aminoacyl-tRNA synthetaseactivities of a bacterial host broadens the scope of proteinengineering, by permitting production of natural and artificialproteins, with novel chemical and physical properties.

1. A method for producing a modified polypeptide, comprising: a.providing a host cell, the host cell comprising: i. a vector having apolynucleotide sequence encoding an aminoacyl-tRNA synthetase for anamino acid analogue; and ii. a vector having a polynucleotide sequenceencoding a polypeptide molecule of interest so as to produce a hostvector system; wherein the vectors of (i) and (ii) may be the same ordifferent b. growing the host cell in a medium so that the host vectorsystem expresses the aminoacyl-tRNA synthetase c. replacing the mediumwith a medium which has the desired amino acid analogue or adding thedesired amino acid analogue to the medium d. growing the host cell inthe medium which has the desired amino acid analogue under conditions sothat the host cell expresses the polypeptide molecule of interest andthe desired amino acid analogue is incorporated into the polypeptidemolecule of interest thereby producing the modified polypeptide.
 2. Themethod of claim 1 wherein the vector having a polynucleotide sequenceencoding an aminoacyl-tRNA synthetase and the vector having apolynucleotide sequence encoding a polypeptide of interest are the samevector.
 3. The method of claim 1 wherein the host cell is an auxotrophichost cell.
 4. The method of claim 3 wherein the auxotrophic host cell isfrom an organism that is selected from the group consisting of:bacteria, yeast, mammal, insect, and plant.
 5. The method of claim 1wherein the polynucleotide sequence encoding an aminoacyl-tRNAsynthetase originated from a different cell than the host cell.
 6. Amodified polypeptide produced by the method of any one of claims 1-5. 7.A recombinant vector comprising a polynucleotide sequence encoding anaminoacyl-tRNA synthetase for an amino acid analogue and apolynucleotide sequence encoding a polypeptide molecule of interest. 8.The vector of claim 7 further comprising at least one expressionelement.
 9. The vector of claim 8 wherein at least one expressionelement is selected from the group consisting of: promoter sequence,secretion signal, enhancer sequence, transcription terminator,Shine-Dalgarno sequence, initiator codon, and termination codon.
 10. Thevector of claim 7 wherein the polynucleotide sequence encoding anaminoacyl-tRNA synthetase originated from a different cell than the hostcell.
 11. A composition comprising the vector of claim 7, and a hostcell.
 12. The composition of claim 11 wherein the host cell is anauxotrophic host cell.
 13. The composition of claim 12 wherein theauxotrophic host cell is from an organism that is selected from thegroup consisting of: bacteria, yeast, mammal, insect, and plant.